Luminex Based Genetic and Protein Assay Development Presentation

Luminex Based Assay Development

Stephen Angeloni PhD Sr Field Application Scientist Luminex Corporation

Luminex Technology A Broad Range of Applications

2 | copy Copyright 2012 Luminex Corporation Confidential

Research

Human Diagnostics

Animal Health

Ag Sciences

Food Safety

Biodefense

Environmental Monitoring

Escondidorsquos

Escondidorsquos

Luminex Corporate Offices Global Presence



Luminex Global Presence Over 11000 instruments shipped globally

Luminex Propelling Knowledge


Over 24000 peer-reviewed scientific studies and growing For complete listing see wwwluminexcorpcompublications

Luminex Partners Over 60 partners worldwide


Luminex Diagnostics More than 60 US FDA 510(k) Cleared Platforms


xMAP Technology An Overview


xMAP Technology Microsphere-based Arrays

MagPlextrade

Superparamagnetic microspheres 65 um

Microsphere Advantages

bull Fast liquid phase kinetics

bull Proprietary dyeing process

bull Unique bead signatures

bull High surface-to-volume ratio

bull Surface carboxyl groups

MicroPlextrade

Microspheres 56 um

xMAP Applications An Incredibly Flexible Platform

Bead sets can be coated with reagents specific to many bioassays


Proteins Nucleic Acids

bull Immunoassays raquo Sandwich Capture raquo Multiplex ELISA

bull Receptor-LigandProtein interaction Assays

bull Enzyme Substrate

bull Gene Expression bull microRNA Profiling bull Genotyping

raquoSNP raquoCNV raquoSequence Detection

Excite One Excitation Wavelength

Observe Three Emission Wavelengths


Spectral Addressing Multiplexing to 500 AnalytesWell with MagPlex beads


Spectral Addressing = Multiplexing Each Bead Associated with a Specific Analyte in each reaction

With each bead color assigned an analyte each reaction measures multiple targets for any genomic or proteomic assay


Several Advantages to Multiplexing Use less sample less reagents get more data faster get more accurate data

A 107 plex Gene Expression assay Luminex

14 samples in triplicate Done in 42 wells of a 96 well

plate

Single plex chemistry like TaqMan or another real time

PCR chemistry would required 4494 wells = 468 96 well

plates

24 plex Protein assay Our partners make protein assays that can analyze as

many as 43 proteins or more with some kits Running a

24 plex assay on 27 samples in triplicate required 81 wells

of a 96 well plate

A standard single plex ELISA chemistry would require

1944 wells and more than 20 96 well plates If 43 marker

assay single plex would need 3483 reactions or 363 96

well plates

Single Plex Reactions

Luminex Multiplexing

Real Examples

Arrays

And

NGS

RT-PCR


Discovery

amp

Validation-1

An

aly

tes

S

am

ple

Samples Day

10s 100s 1000s 10000s

10

100

1000

10000 3D Throughput

1 plate = 384 samples

500 plex = 192000 dpplate

6 x 384 platesday

2304 samplesday

1152000 dpday

Luminex Instruments Unique Combination of Flexibility Multiplexing and Throughput

MAGPIX

50 Plex

LX200

80-100 Plex

FLEXMAP 3D

500 Plex

Flow Cytometry and Imaging Comparison

Identify bead region

based on internal dye

concentrations

Quantify binding

events

Interrogate bead

with red laser

(635 nm)

Interrogate label

with green laser

(525 nm)

Sheath Fluid

Flow Cytometry-Based Analysis ndash Luminex 200 amp FLEXMAP 3D

LEDImage-Based Analysis - MAGPIX Interrogate label with

green LED (525 nm)

Interrogate bead with red

LED (635 nm)

Identify and quantify with

CCD imager

Beads in Chamber Magnetic

Capture

16 | copy Copyright 2012 Luminex Corporation

Genomic Assays

Direct Hybridization

bull Couple sequence-specific C18 spacer amine-modified oligo to bead

bull Probes are matched for length ~20bp for SNPs 50-70bp okay for unrelated sequences and mismatches are centered or distributed


19 | copy Copyright 2012 Luminex Corporation 19 | copy Copyright 2013 Luminex Corporation

Sequence-specific Oligo

bull Label target nucleic acid sequence to be detected with biotin PCR primer bull Amplicons should be le 300bp if possible bull Denature hybridize to beads incubate with SAPE and detect



Detection

SAPE

DDLGSK HPV Genotyping Assay


Luminex assays Developed and Published by DDL

Stability study on several assays 2yr 3 lots 4degC

Target Multiplex Intended use Reference

HPV (high-risk

mucosal anogenital)

21-plex Research Geraets et al 2009

Chlamydia trachomatis

16-plex Research amp Epidemiology

Quint et al 2009

HPV (wart) 25-plex Research amp Epidemiology

De Koning et al 2010

HPV (high-risk

mucosal anogenital)


Van Alewijk et al 2013

HPV (mucosal

anogenital)


Kleter et al to be submitted

DDL is a contract research organization and is available for collaborations and service


xMAPreg Salmonella Serotyping Assay RUO Kit Training


bull BADGE Beads Array for the Detection of Gene Expression a high-throughput diagnostic bioassay Yang L DK Tran and X Wang Genome Res 2001 11(11) p 1888-98

Direct Hybridization BADGE Gene Expression Assay


Detection

RNA

Biotinylated cDNA

Direct Hybridization Requires optimization of coupled capture oligonucleotides


xTAG Technology Simplifying Nucleic Acid Assays


bull Proprietary sets of 24 base oligonucleotides are coupled to xTAG beads

bull Each xTAG bead regionrsquos tag is complementary to a specific tag sequence engineered onto reporter molecules

bull This approach as been validated and used in multiplexed clinical genetic tests

1 2

TAG-1 Sequence A TAG-2 Sequence B


Key to using xTAG Technology For Simplifying Nucleic Acid Assays

MagPlex-TAG Bead Mix

1

2

TAG-1

TAG-2


Using xTAG Technology For Different types of Nucleic Acid Assays

o Gene expression

o SNP typing Specific sequence detection

o Copy Number Variation Analysis (CNV)

o miRNA analysis

o And variations of these chemistries for a number of

applications


Allele Specific Primer Extension LMA (OLA) Target Specific PCR

xTAG Chemistries For Different DNA Analysis Assays

ANNEAL

Ligate

PCR

TAG1

TAG2

cDNA or

gDNAPCR

HYBRIDIZE

amp

LABEL

dA

dG dT

dC -

ANNEAL

amp

Extend

TAG1

TAG2

HYBRIDIZE

amp

LABEL

gDNAPCR

SNP typing CF

CYP 450 RVP

Gene expression SNP typing

CNV etc

RVP FAST GPP FAST

HYBRIDIZE

amp

LABEL

TAG1

TAG2 ANNEAL

amp

PCR

gDNA

miRNA Analysis Nuclease Protection Approach

30

Step-down Probe Hybridization ndash DNARNA chimeric probes hybridize

to target miRNAs during incremental reductions in annealing temperature

2 hours

RNase Digestion ndash Excess probes single-stranded RNAs and mismatched

probes are digested Only perfectly-matched probes are protected

30 minutes

SAPE Incubation ndash A brief incubation with streptavidin-conjugated

R-Phycoerythrin (SAPE) incorporates reporter molecules

30 minutes

Detection ndashTargets of interest are quantified on a Luminex instrument

lt 5 hours total to results

Microsphere Hybridization ndash miRNA-chimeric probe complexes are

hybridized to microspheres

30 minutes

LMA Multiplex Gene Expression Assays

cDNA required for LMA Gene Expression Assay


o Oligo-dT primed cDNA synthesis Can generate cDNA only from poly-A tailed mRNAs

Can be optimized with poly-A capture methods

Can be more sensitive than TaqMan and other PCR

methods

o Random primed cDNA synthesis Can generate cDNA from all RNA species

Can also detect non-ploy-A transcripts

Slightly shorter protocol than poly-A capture method

LMA = Ligation Mediated Amplification

LMA Poly-A mRNA Capture Approach


TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

AAAAAAA RNA

AAAAAAA RNA

AAAAAAA RNA

bull Add sample raquoCell lysates

raquoPurified Total RNA bull Polyadenylated RNA is captured with immobilized poly-T

bull Unbound materials are washed away

LMA Poly-A mRNA Capture Approach cDNA Synthesis


TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

AAAAAAA RNA

AAAAAAA RNA

AAAAAAA RNA

cDNA

cDNA

cDNA

bull RT Synthesis of 1st strand cDNA

LMA Poly-A mRNA Capture Approach Probe Hybridization


TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

cDNA

cDNA

cDNA

bull Probes added raquo2 probes per transcript

raquo~25 bases of target-specific sequence per probe

bull Overnight or shorter Hybridization

LMA Probe Design


Downstream LMA Probe

Upstream LMA Probe

cDNA

~50bp Sequence Specific Region

Universal Primer 1

5rsquo 3rsquo

LMA Poly-A mRNA Capture Approach PCR Amplification


bull Same universal primers used for all probe sets

bull No size bias all PCR products are same size

bull Downstream primer is biotinylated

LMA Poly-A mRNA Capture Approach Bead Hybridization and Detection


bull PCR products are λExo treated and hybridized to MagPlex-TAG beads

bull Biotinylated products incubated with Streptavidin-Phycoerythrin (SAPE)

bull Detection on Luminex Instruments


TAG

SAPE

Detection

LMA Poly-A mRNA Capture Approach Workflow


lt3 Hours Setup

lt7 Hours to Detection


Literature References

Peck et al 2006 A method for high-throughput gene expression signature analysis Haining et al 2008 High-throughput gene expression profiling of memory differentiation in primary human T cells Shao X J et al 2011 (Chinese) Development of a Bead-based Liquid Array for Analysis of Gene Expression Profiling Reijans M et al 2008 RespiFinder a new multi parameter test to differentially identify fifteen respiratory viruses Used for L1000 platform by GENOMETRY INC

Luminex Partner Options for Genomic assays


Luminex Based Multiplex SNP Genotyping Assays


Different SNP Chemistries

LMA (OLA) Allele Specific Primer Extension

ANNEAL

Ligate

PCR

TAG1

TAG2

cDNA or

gDNAPCR

HYBRIDIZE

amp

LABEL

LMA Genotyping Assay


LMA Probe Design akahellip LMA MLPA MolPCR


Upstream LMA Probes

T

Universal Primer 1

5rsquo

A

5rsquo

G


Ligation Probe Mix Purified Genomic DNA

SNP

T

LMA Single Nucleotide Analysis

Hybridize Multiplex Ligation Probes

T

T


PCR Amplification

UP 1

P

No Amplification

B-UP 2

Process Amplicons for Analysis


Universal Prime mix

UP 1

P

B-UP 2

UP 1

P

B-UP 2

Ligate

T T

Bead Hybridization and Detection


bull PCR products are λExo treated hybridized to MagPlex-TAG beads




TAG

SAPE

Detection

LMA Genotyping Workflow

PCR Signal Amp 25 hours

Bead Hyb 80 minutes

Probe Ligation 70 minutes

Detection 20-75 Minutes

bull Overnight or shorter Incubation Followed by Data Collection

bull Automation-Friendly Workflow

bull PCR Uses Universal Primers for Unbiased Signal Amplification bull Optimized for Fast Read Times

Probe Hybridization Overnight or less

Incubation



Kaderali et al 2003 Primer-design for multiplexed genotyping Zhi J and E Hatchwell 2008 Human MLPA Probe Design (H-MAPD) a probe design tool for bothelectrophoresis-based and bead-coupled human multiplexligation-dependent probe amplification assays Milosevic et al 2010 Development and validation of a comprehensive mutation and deletion detection assay for SDHB SDHC and SDHD Deshpande et al 2010 A rapid multiplex assay for nucleic acid-based diagnostics Song et al 2010 Simultaneous Pathogen Detection and Antibiotic Resistance Characterization Using SNP-Based Multiplexed Oligonucleotide Ligation-PCR (MOL-PCR) Stucki et al 2012 Two new rapid SNP-typing methods for classifying Mycobacterium tuberculosis complex into the main phylogenetic lineages Deshpande amp White 2012 Multiplexed nucleic acid-based assays for molecular diagnostics of human disease

OLA (LDR-FMA) Genotyping Assay


OLA (LDR-FMA) Genotyping OLA = Oligo Ligation Amplification LDR-FMA = ligase detection reaction - fluorescent microsphere assay

Generate ligation probe target regions of interest with PCR

Targeted PCR


OLA (LDR-FMA) Probe Design

Downstream LDR Probe

Upstream LDR Probes

A T

G


Ligation Probe Mix Amplified Genomic Target

SNP

A

A

OLA Single Nucleotide Analysis


A


Multiple

Ligation

cycles

Process Ligated Probes for Analysis




bull hybridized to MagPlex-TAG beads




TAG

SAPE

Detection


Carnevale et al 2007 A Multiplex Ligase Detection Reaction-Fluorescent Microsphere Assay for Simultaneous Detection of Single Nucleotide Polymorphisms Associated with Plasmodium falciparum Drug Resistance Bruse et al 2008 Improvements to bead-based oligonucleotide ligation SNP genotyping assays Mehlotra et al 2011 Chemokine (C-C motif) receptor 5 -2459 genotype in patients receiving highly active antiretroviral therapy race-specific influence on virologic success Henry-Halldin et al 2012 Multiplex Assay for Species Identification and Monitoring of Insecticide Resistance in Anopheles punctulatus Group Populations of Papua New Guinea


ASPE SNP Typing Assays


Forward primers

gDNA

Sequence Specific Region

5rsquo

3rsquo

ASPE Probe Design With one Biotinylated-Nucleotide Chemistry

A

T

5rsquo

3rsquo

G

dATP dTTP dGTP dCTP dCTP


ASPE SNP Genotyping gDNA Target Amplification

Amplify probe target regions of interest with PCR

Targeted PCR


ASPE SNP Detection Primer Extension on amplified regions

A

5rsquo

3rsquo

G

5rsquo

3rsquo

T




A

5rsquo

3rsquo

G 5rsquo

3rsquo

T



ASPE SNP Detection Primer with complimentary base is extended and biotynalated


bull PCR products hybridized to MagPlex-TAG beads



xTAG


Detection

SAPE

ASPE SNP Detection With Biotinylated-nucleotide


Strom et al 2005 Technical validation of a multiplex platform to detect thirty mutations in eight genetic diseases prevalent in individuals of Ashkenazi Jewish descent Koo et al 2007 Multiplexed genotyping of ABC transporter polymorphisms with the Bioplex suspension array Li et al 2011 A novel liquidchip platform for simultaneous detection of 70 alleles of DNA somatic mutations on EGFR KRAS BRAF and PIK3CA from formalin-fixed and paraffin-embedded slides containing tumor tissue


Target Specific PCR Chemistry


Target Specific PCR TS-PCR Chemistry

TS PCR Probe Design With Biotinylated primer and standard nucleotides


xTAG primer

gDNA


Biotinylated primer

5rsquo 3rsquo

3rsquo

dATP dTTP dGTP dCTP

= Carbon Spacer


TAG primer Biotinylated primer

TS PCR Probe Design With Multiplex Probe mix

Multiplex Amplification Products







Detection

Sequence Detection

TAG

o Easy and Fast bull Simple sample processing from a number of different sample types such as

swabs stool cultures etc

bull Results in a few hours

o Used in Diagnostics for bull Detection of 8 viruses and subtypes of Influenza A Influenza A subtype H1

Influenza A subtype H3 Influenza B Respiratory Syncytial Virus (RSV) Human Metapneumovirus (hMPV) Rhinovirus and Adenovirus

bull Detection of 11 gastrointestinal pathogens and subtypes of bacteria viruses and parasites of C difficile norovirus E coli Salmonella rotavirus A Campylobacter and Shigella

FAST PCR Assay Characteristics Summary


Multiplex microRNA Assay Nuclease protection Approach

bull Extremely Specific Results raquoCan discriminate single base mismatches anywhere in

mature miRNA sequences

bull Fast Results raquoFrom total RNA to results in less than 5 hours with less than

30 minutes of handling

bull Unbiased Sensitivity raquoNo amplification or sample labeling required yet data from

50-500ng total RNA with single nucleotide difference resolution

Multiplex miRNA Nuclease protection Assay Characteristics


Workflow

75



2 hours



30 minutes



30 minutes





30 minutes

76

hsa-let-7a UGAGGUAGUAGGUUGUAUAGUU

hsa-let-7c UGAGGUAGUAGGUUGUAUGGUU

Specificity Single Base Mismatch Discrimination

Discrimination of single

base mismatch near ends

0

500

1000

1500

2000

2500

3000

3500

4000

BG A - 02fmol B - 02fmol

Sample

Sig

nal In

ten

sit

y (

MF

I)

let-7c

Let-7a

77

VAL0538 - Attachment 3

miR-107 vs miR-103

0

1000

2000

3000

4000

5000

6000


Sample

Sig

na

l In

ten

sit

y (

MF

I)

miR-103

miR-107

hsa-miR-107 AGCAGCAUUGUACAGGGCUAUCA

hsa-miR-103 AGCAGCAUUGUACAGGGCUAUGA


hsa-miR-103 AGCAGCAUUGUACAGGGCUAUGA Discrimination of single



Specificity for Targets in Complex mix Closely-Related miRNAs in 60 Plex

X-Hybe - target Input at 10fmol

0

1000

2000

3000

4000

5000

6000

7000

8000

9000

10000

hsa-m

iR-1

07

hsa-m

iR-1

47

hsa-m

iR-1

81a

hsa-m

iR-1

92

hsa-m

iR-1

9b

hsa-m

iR-2

20a

hsa-m

iR-2

1hsa-m

iR-2

18

hsa-le

t-7c

hsa-m

iR-1

00

hsa-m

iR-1

25b

hsa-m

iR-

hsa-m

iR-2

05

hsa-m

iR-2

21

hsa-m

iR-2

00a

hsa-m

iR-2

00c

hsa-m

iR-2

06

hsa-m

iR-1

0a

hsa-m

iR-1

6

hsa-m

iR-9

hsa-m

iR-4

24

hsa-m

iR-2

0a

hsa-m

iR-2

0b

hsa-le

t-7a

hsa-m

iR-9

5hsa-m

iR-1

36

hsa-m

iR-1

95

hsa-m

iR-2

10

hsa-m

iR-2

12

hsa-m

iR-

hsa-m

iR-1

7

hsa-m

iR-1

22

hsa-m

iR-1

43

hsa-m

iR-1

45

hsa-m

iR-1

06a

hsa-m

iR-

hsa-m

iR-1

0b

hsa-m

iR-1

hsa-m

iR-2

23

hsa-m

iR-1

81b

hsa-m

iR-1

55

hsa-le

t-7g

hsa-le

t-7i

hsa-m

iR-9

6

hsa-m

iR-1

37

hsa-m

iR-1

85

hsa-m

iR-1

38

hsa-m

iR-1

98

hsa-m

iR-1

33a

hsa-m

iR-1

33b

hsa-m

iR-1

5a

hsa-m

iR-3

4a

hsa-m

iR-2

9a

hsa-m

iR-2

9b

hsa-m

iR-1

03

hsa-m

iR-2

22

hsa-m

iR-1

46a

cel-m

iR-3

9cel-m

iR-5

4

cel-m

iR-2

38

Regions

Ne

t S

ign

al In

ten

sit

y (

MF

I)

106a

17

10 fmol input miR-106a CAAAGUGCUAACAGUGCAGGUAG miR-17 CAAAGUGCUUACAGUGCAGGUAG

Total RNA Samples

53-Plex Assay

miR-9 miR-122 miR-1

0

5000

10000

15000

20000

25000

Samples

Ne

t S

ign

al In

ten

sit

y (

MF

I)

miR-9

miR-122

miR-1

Specificity in Tissue Samples Correct Detection of Tissue-Specific miRNAs

miRNAs are specifically

detected in correct tissues

Sensitivity in Complex mix 200 amol Molecules or Less ndash Unamplified

Dose Response - 53 Synthetic Oligos

289704

12431797

4050

7044

12899

18030

24973

23884

21852

19249

11326

6573

3852

2471

0

5000

10000

15000

20000

25000

30000

002fmol 0039fmol 008fmol 0156fmol 0313fmol 0625fmol 125fmol 2fmol

Input Amount

Ne

t S

ign

al In

ten

sit

y (

MF

I)

200 amol

53 Multiplexed Synthetic Oligos on LX200

81

Sensitivity limits Titration below 100 amols ndash Unamplified 53 plex Synthetic Oligos on FlexMAP 3D

0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000

0025fmol 005fmol 01fmol

Input Amount (fmol) (Note Expanded Scale)

Ne

t S

ign

al in

ten

sit

y (

MF

I)

82

miR-133a

000

020

040

060

080

100

120

brain breast cervix heart liver lung prostate thyroid

Total RNA Samples

Rela

tive C

han

ge t

o H

eart

FlexmiR v2

qRT-PCR

Tissue

Re

lati

ve

Ch

an

ge

Accuracy vs qPCR Correlation to qRT-PCR for miR-133a

LNP-miRNA

qRT-PCR

83

miRNA

Re

lati

ve

Ch

an

ge

Accuracy vs qPCR Correlation to qPCR of 6 microRNAs Fold Change of Tumor vs NAT - Lymphoma Samples

TumorNAT - Lymphoma Samples

0

5

10

15

20

25

miR-21 miR-143 miR-155 miR-146a miR-145 miR-16

miRNA

Rela

tive C

han

ge

FlexmiR v2

qRT-PCR

LNP-miRNA

qPCR

84

Accuracy at Different RNA Inputs Consistent Results with Varying Total Prostate RNA

Input Titration - Prostate Total RNA

0

5000

10000

15000

20000

25000

30000

hsa-m

iR-1

07

hsa-m

iR-1

47

hsa-m

iR-1

81a

hsa-m

iR-1

92

hsa-m

iR-1

9b

hsa-m

iR-2

20a

hsa-m

iR-2

1hsa-m

iR-2

18

hsa-le

t-7c

hsa-m

iR-1

00

hsa-m

iR-1

25b

hsa-m

iR-

hsa-m

iR-2

05

hsa-m

iR-2

21

hsa-m

iR-2

00a

hsa-m

iR-2

00c

hsa-m

iR-2

06

hsa-m

iR-1

0a

hsa-m

iR-1

6hsa-m

iR-9

hsa-m

iR-4

24

hsa-m

iR-2

0a

hsa-m

iR-2

0b

hsa-le

t-7a

hsa-m

iR-9

5hsa-m

iR-1

36

hsa-m

iR-1

95

hsa-m

iR-2

10

hsa-m

iR-2

12

hsa-m

iR-

hsa-m

iR-1

7hsa-m

iR-1

22

hsa-m

iR-1

43

hsa-m

iR-1

45

hsa-m

ir-1

06a

hsa-m

iR-

hsa-m

iR-1

0b

hsa-m

iR-1

hsa-m

iR-2

23

hsa-m

iR-1

81b

hsa-le

t-155

hsa-le

t-7g

hsa-m

iR-7

ihsa-m

iR-9

6hsa-m

iR-1

37

hsa-m

iR-1

85

hsa-m

iR-1

38

hsa-m

iR-1

33a

hsa-m

iR-1

33b

hsa-m

iR-1

5a

hsa-m

iR-3

4a

hsa-m

iR-2

9a

hsa-m

iR-2

9b

hsa-m

iR-1

03

hsa-m

iR-2

22

hsa-m

iR-1

46a

hsa-m

iR-3

9cel-m

iR-3

9cel-m

iR-2

38

Regions

Ne

t S

ign

al in

ten

sit

y (

MF

I)

50ng

100ng

250ng

500ng

bull Fast Easy and Unbiased raquoOnly 30 Minutes of Handling

raquoNo Sample Labeling or Amplification

raquoTime to Results lt5 hours

bull Solid Performance

raquoReproducible CVs lt15

raquoSensitive 50-500ng Total RNA Input

raquoHighly Specific Discriminates Single Base Mismatches

bull Flexible

raquoMultiplexing of gt80 miRNAswell possible

raquoDesign your own custom probes

miRNA Assay Characteristics Summary


Proteomic Assays



Nucleic Acids



Multiplex Serological (ELISA) Assays


Multiplex ELISA Mix beads with sera

Multiplex ELISA Mix Beads with Biotinylated Detection Antibody mix

Biotin labeled anti IgG (1 to 4) IgA

or IgM

Multiplex ELISA Mix Beads with SA-PE

Can now determine if antibody response to antigen

is IgG1 2 3 4 or IgA or IgM


Multiplex Detection

Detection

bull Each well can be plexed to measure as many antigens as reagents will allow up to 500 maximum

IgG

1 2 3 4



Anderson S P Wakeley G Wibberley K Webster and J Sawyer (2011) Development and

evaluation of a Luminex multiplex serology assay to detect antibodies to bovine herpes virus 1

parainfluenza 3 virus bovine viral diarrhoea virus and bovine respiratory syncytial virus with

comparison to existing ELISA detection methods Journal Of Immunological Methods 366 79-88

Gimenez-Lirola L G Y-H Jiang D Sun H Hoang K-J Yoon P G Halbur and T Opriessnig

(2014) Simultaneous Detection of Antibodies against Apx Toxins ApxI ApxII ApxIII and ApxIV in

Pigs with Known and Unknown Actinobacillus pleuropneumoniae Exposure Using a Multiplexing

Liquid Array Platform Clinical and Vaccine Immunology 21(1) 85 - 95

Langenhorst R J S Lawson A Kittawornrat J J Zimmerman Z Sun Y Li J Christopher-

Hennings E A Nelson and Y Fang (2012) Development of a fluorescent microsphere immunoassay

for detection of antibodies against porcine reproductive and respiratory syndrome virus using oral fluid

samples as an alternative to serum-based assays Clin Vaccine Immunol 19(2) 180-189

Smits G P P G van Gageldonk L M Schouls F R M van der Klis and G A M Berbers (2012)

Development of a Bead-Based Multiplex Immunoassay for Simultaneous Quantitative Detection of

IgG Serum Antibodies against Measles Mumps Rubella and Varicella-Zoster Virus Clinical And

Vaccine Immunology 19(3) 396-400


Multiplex Antigen Specific Ig ELISA assay Beads Coated with Capture Antibodies for Different Ig types

Y Y

Y

IgG1 IgG2

IgG3

IgG4 IgA


Multiplex Antigen Specific Ig ELISA assay Beads with Capture Antibodies incubated with serum sample

Y Y

Y


Multiplex Antigen Specific Ig ELISA assay Beads Capture Antibodies of Different Ig types

Y Y

Y


Multiplex Antigen Specific Ig ELISA assay Beads Incubated with Biotin Labeled Antigen

Y Y

Y


Multiplex Antigen Specific Ig ELISA assay Different Ig types will bind Antigen

Y Y

Y


Multiplex Antigen Specific Ig ELISA assay Incubate with SAPE (Streptavidin-R-Phycoerythrin)

Y Y

Y


Multiplex Antigen Specific Ig ELISA assay Detection

Detection

bull Each well can be plexed to measure as many antigen specific Ig subtypes as needed

bull Allows more rapid analysis of Ig type responses to multiple antigens in large numbers of samples

Antigen

1 2 3

Multiplex Sandwich Assays


Multiplex Sandwich Assays Beads Coated with Capture Antibodies Mixed with Sample


Multiplex Sandwich Assays Targets Bind to Capture Antibodies


Multiplex Sandwich Assays Mix with Labeled Target Specific Antibodies

Y Y Y

Y

Y

Y Y

Y Y

Y Y

Y



Y


Detection

Multiplex Detection




Bjerre M T Hansen A Flyvbjerg and E Tonnesen (2009) Simultaneous

detection of porcine cytokines by multiplex analysis Development of magnetic

bioplex assay Veterinary Immunology And Immunopathology 130 53-58

Sun K Q Wang X Huang M Zhen W Li and L Zhang (2007) Establishment

of multiplexed microsphere-based flow cytometric assay for multiple human tumor

markers Acta Pharmacologica Sinica 28(12) 2011-2018

Protein Interaction and Receptor LigandSubatrate Assays


Types of Receptor Ligand and Protein Interaction Assays

Types of Receptor Ligand Interaction assays




Inhibition by different

compounds



Types of Protein Interaction Assays


compounds


Multiplex Enzyme Substrate Interaction Assays A two plex assay Labeled Substrate

SAPE SAPE


= inhibiting compound

Multiplex Enzyme Substrate Interaction Assays A two plex assay Inhibitor Treatment


Multiplex Enzyme Substrate Interaction Assays Inhibitor Treatment at Different Concentrations


Multiplex Enzyme Substrate Interaction Assays Add Labeled Substrate


Multiplex Enzyme Substrate Interaction Assays Labeled Substrate competes with Inhibitor


Multiplex Enzyme Substrate Interaction Assays Wash out Excess Inhibitor and Labeled Substrate


Multiplex Enzyme Substrate Interaction Assays Hybridize with SAPE

SAPE SAPE


Detection

Multiplex Detection

bull Each well can be plexed to measure as many antigens as reagents will allow up to 500 maximumreaction



Bachovchin D A et al (2014) A high-throughput multiplexed assay for superfamily-wide profiling

of enzyme activity Nature Chemical Biology 10(8) 656 - 666

Poetz O K Luckert T Herget and T Joos (2009) Microsphere-based co-immunoprecipitation in

multiplex Analytical Biochemistry 395(2) 244-248

Blazer L L D L Roman M R Muxlow and R R Neubig (2010) Use of flow cytometric methods

to quantify protein-protein interactions Curr Protoc Cytom Chapter 13 Unit 13 11 11-15

Rimmele S P Gierschik T Joos and N Schneiderhan-Marra (2010) Bead-based protein-protein

interaction assays for the analysis of Rho GTPase signaling Journal Of Molecular Recognition 23(6)

543-550

Schmohl M S Rimmele O Poetz Y Kloog P Gierschik T Joos and N Schneiderhan-Marra (2010)

Protein-protein-interactions in a multiplexed miniaturized format a functional analysis of Rho GTPase

activation and inhibition Proteomics 10(8) 1716-1720

Schmohl M S Rimmele P Gierschik T Joos and N Schneiderhan-Marra (2012) Functional

Analysis of Rho GTPase Activation and Inhibition in a Bead-Based Miniaturized Format Rho

GTPases 827 271-282

Resources for Developing Multiplex Assays



1 The Kit Finder Allows users to search for existing kits made by Luminex partners

3 The xMAP Cookbook Helps users develop their own assays with all of their own reagents

2 Papers with protocols of interest Try the Luminex publication search tool



The xMAP Cookbook is a collection of easy to follow protocols with lists of reagents and equipment needed to develop you own Luminex based assay

Interested in Getting Started

And visit us on the web at wwwluminexcorpcom



Single Plex Assays

To Luminex

Multiplex

Assays

Thank you for Attending

I will now take questions about converting yourhellip

Luminex Technology A Broad Range of Applications


Research

Human Diagnostics

Animal Health

Ag Sciences

Food Safety

Biodefense

Environmental Monitoring

Escondidorsquos

Escondidorsquos















MagPlextrade








MicroPlextrade

Microspheres 56 um





















plate



plates




of a 96 well plate




well plates



Real Examples

Arrays

And

NGS

RT-PCR


Discovery

amp

Validation-1

An

aly

tes

S

am

ple

Samples Day

10s 100s 1000s 10000s

10

100

1000

10000 3D Throughput



6 x 384 platesday

2304 samplesday

1152000 dpday


MAGPIX

50 Plex

LX200

80-100 Plex

FLEXMAP 3D

500 Plex




concentrations

Quantify binding

events

Interrogate bead

with red laser

(635 nm)

Interrogate label

with green laser

(525 nm)

Sheath Fluid



green LED (525 nm)


LED (635 nm)


CCD imager


Capture


Genomic Assays










Detection

SAPE






HPV (high-risk

mucosal anogenital)




Quint et al 2009



HPV (high-risk

mucosal anogenital)



HPV (mucosal

anogenital)










Detection

RNA

Biotinylated cDNA








1 2





1

2

TAG-1

TAG-2



o Gene expression



o miRNA analysis


applications




ANNEAL

Ligate

PCR

TAG1

TAG2

cDNA or

gDNAPCR

HYBRIDIZE

amp

LABEL

dA

dG dT

dC -

ANNEAL

amp

Extend

TAG1

TAG2

HYBRIDIZE

amp

LABEL

gDNAPCR

SNP typing CF

CYP 450 RVP


CNV etc

RVP FAST GPP FAST

HYBRIDIZE

amp

LABEL

TAG1

TAG2 ANNEAL

amp

PCR

gDNA


30



2 hours



30 minutes



30 minutes





30 minutes







methods







TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

AAAAAAA RNA

AAAAAAA RNA

AAAAAAA RNA






TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

AAAAAAA RNA

AAAAAAA RNA

AAAAAAA RNA

cDNA

cDNA

cDNA




TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

cDNA

cDNA

cDNA




LMA Probe Design



Upstream LMA Probe

cDNA


Universal Primer 1

5rsquo 3rsquo












TAG

SAPE

Detection



lt3 Hours Setup











ANNEAL

Ligate

PCR

TAG1

TAG2

cDNA or

gDNAPCR

HYBRIDIZE

amp

LABEL





Upstream LMA Probes

T

Universal Primer 1

5rsquo

A

5rsquo

G



SNP

T



T

T


PCR Amplification

UP 1

P

No Amplification

B-UP 2



Universal Prime mix

UP 1

P

B-UP 2

UP 1

P

B-UP 2

Ligate

T T







TAG

SAPE

Detection



Bead Hyb 80 minutes







Incubation








Targeted PCR




Upstream LDR Probes

A T

G



SNP

A

A



A


Multiple

Ligation

cycles









TAG

SAPE

Detection






Forward primers

gDNA


5rsquo

3rsquo


A

T

5rsquo

3rsquo

G





Targeted PCR



A

5rsquo

3rsquo

G

5rsquo

3rsquo

T




A

5rsquo

3rsquo

G 5rsquo

3rsquo

T








xTAG


Detection

SAPE










xTAG primer

gDNA


Biotinylated primer

5rsquo 3rsquo

3rsquo

dATP dTTP dGTP dCTP

= Carbon Spacer











Detection

Sequence Detection

TAG


















Workflow

75



2 hours



30 minutes



30 minutes





30 minutes

76






0

500

1000

1500

2000

2500

3000

3500

4000


Sample

Sig

nal In

ten

sit

y (

MF

I)

let-7c

Let-7a

77


miR-107 vs miR-103

0

1000

2000

3000

4000

5000

6000


Sample

Sig

na

l In

ten

sit

y (

MF

I)

miR-103

miR-107









0

1000

2000

3000

4000

5000

6000

7000

8000

9000

10000

hsa-m

iR-1

07

hsa-m

iR-1

47

hsa-m

iR-1

81a

hsa-m

iR-1

92

hsa-m

iR-1

9b

hsa-m

iR-2

20a

hsa-m

iR-2

1hsa-m

iR-2

18

hsa-le

t-7c

hsa-m

iR-1

00

hsa-m

iR-1

25b

hsa-m

iR-

hsa-m

iR-2

05

hsa-m

iR-2

21

hsa-m

iR-2

00a

hsa-m

iR-2

00c

hsa-m

iR-2

06

hsa-m

iR-1

0a

hsa-m

iR-1

6

hsa-m

iR-9

hsa-m

iR-4

24

hsa-m

iR-2

0a

hsa-m

iR-2

0b

hsa-le

t-7a

hsa-m

iR-9

5hsa-m

iR-1

36

hsa-m

iR-1

95

hsa-m

iR-2

10

hsa-m

iR-2

12

hsa-m

iR-

hsa-m

iR-1

7

hsa-m

iR-1

22

hsa-m

iR-1

43

hsa-m

iR-1

45

hsa-m

iR-1

06a

hsa-m

iR-

hsa-m

iR-1

0b

hsa-m

iR-1

hsa-m

iR-2

23

hsa-m

iR-1

81b

hsa-m

iR-1

55

hsa-le

t-7g

hsa-le

t-7i

hsa-m

iR-9

6

hsa-m

iR-1

37

hsa-m

iR-1

85

hsa-m

iR-1

38

hsa-m

iR-1

98

hsa-m

iR-1

33a

hsa-m

iR-1

33b

hsa-m

iR-1

5a

hsa-m

iR-3

4a

hsa-m

iR-2

9a

hsa-m

iR-2

9b

hsa-m

iR-1

03

hsa-m

iR-2

22

hsa-m

iR-1

46a

cel-m

iR-3

9cel-m

iR-5

4

cel-m

iR-2

38

Regions

Ne

t S

ign

al In

ten

sit

y (

MF

I)

106a

17


Total RNA Samples

53-Plex Assay

miR-9 miR-122 miR-1

0

5000

10000

15000

20000

25000

Samples

Ne

t S

ign

al In

ten

sit

y (

MF

I)

miR-9

miR-122

miR-1






289704

12431797

4050

7044

12899

18030

24973

23884

21852

19249

11326

6573

3852

2471

0

5000

10000

15000

20000

25000

30000


Input Amount

Ne

t S

ign

al In

ten

sit

y (

MF

I)

200 amol


81


0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000



Ne

t S

ign

al in

ten

sit

y (

MF

I)

82

miR-133a

000

020

040

060

080

100

120


Total RNA Samples

Rela

tive C

han

ge t

o H

eart

FlexmiR v2

qRT-PCR

Tissue

Re

lati

ve

Ch

an

ge


LNP-miRNA

qRT-PCR

83

miRNA

Re

lati

ve

Ch

an

ge



0

5

10

15

20

25


miRNA

Rela

tive C

han

ge

FlexmiR v2

qRT-PCR

LNP-miRNA

qPCR

84



0

5000

10000

15000

20000

25000

30000

hsa-m

iR-1

07

hsa-m

iR-1

47

hsa-m

iR-1

81a

hsa-m

iR-1

92

hsa-m

iR-1

9b

hsa-m

iR-2

20a

hsa-m

iR-2

1hsa-m

iR-2

18

hsa-le

t-7c

hsa-m

iR-1

00

hsa-m

iR-1

25b

hsa-m

iR-

hsa-m

iR-2

05

hsa-m

iR-2

21

hsa-m

iR-2

00a

hsa-m

iR-2

00c

hsa-m

iR-2

06

hsa-m

iR-1

0a

hsa-m

iR-1

6hsa-m

iR-9

hsa-m

iR-4

24

hsa-m

iR-2

0a

hsa-m

iR-2

0b

hsa-le

t-7a

hsa-m

iR-9

5hsa-m

iR-1

36

hsa-m

iR-1

95

hsa-m

iR-2

10

hsa-m

iR-2

12

hsa-m

iR-

hsa-m

iR-1

7hsa-m

iR-1

22

hsa-m

iR-1

43

hsa-m

iR-1

45

hsa-m

ir-1

06a

hsa-m

iR-

hsa-m

iR-1

0b

hsa-m

iR-1

hsa-m

iR-2

23

hsa-m

iR-1

81b

hsa-le

t-155

hsa-le

t-7g

hsa-m

iR-7

ihsa-m

iR-9

6hsa-m

iR-1

37

hsa-m

iR-1

85

hsa-m

iR-1

38

hsa-m

iR-1

33a

hsa-m

iR-1

33b

hsa-m

iR-1

5a

hsa-m

iR-3

4a

hsa-m

iR-2

9a

hsa-m

iR-2

9b

hsa-m

iR-1

03

hsa-m

iR-2

22

hsa-m

iR-1

46a

hsa-m

iR-3

9cel-m

iR-3

9cel-m

iR-2

38

Regions

Ne

t S

ign

al in

ten

sit

y (

MF

I)

50ng

100ng

250ng

500ng








bull Flexible





Proteomic Assays



Nucleic Acids








or IgM





Multiplex Detection

Detection


IgG

1 2 3 4





















Y Y

Y

IgG1 IgG2

IgG3

IgG4 IgA



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Detection



Antigen

1 2 3








Y Y Y

Y

Y

Y Y

Y Y

Y Y

Y



Y


Detection

Multiplex Detection


















compounds





compounds



SAPE SAPE














SAPE SAPE


Detection

Multiplex Detection












543-550






GTPases 827 271-282














Single Plex Assays

To Luminex

Multiplex

Assays



Escondidorsquos

Escondidorsquos















MagPlextrade








MicroPlextrade

Microspheres 56 um





















plate



plates




of a 96 well plate




well plates



Real Examples

Arrays

And

NGS

RT-PCR


Discovery

amp

Validation-1

An

aly

tes

S

am

ple

Samples Day

10s 100s 1000s 10000s

10

100

1000

10000 3D Throughput



6 x 384 platesday

2304 samplesday

1152000 dpday


MAGPIX

50 Plex

LX200

80-100 Plex

FLEXMAP 3D

500 Plex




concentrations

Quantify binding

events

Interrogate bead

with red laser

(635 nm)

Interrogate label

with green laser

(525 nm)

Sheath Fluid



green LED (525 nm)


LED (635 nm)


CCD imager


Capture


Genomic Assays










Detection

SAPE






HPV (high-risk

mucosal anogenital)




Quint et al 2009



HPV (high-risk

mucosal anogenital)



HPV (mucosal

anogenital)










Detection

RNA

Biotinylated cDNA








1 2





1

2

TAG-1

TAG-2



o Gene expression



o miRNA analysis


applications




ANNEAL

Ligate

PCR

TAG1

TAG2

cDNA or

gDNAPCR

HYBRIDIZE

amp

LABEL

dA

dG dT

dC -

ANNEAL

amp

Extend

TAG1

TAG2

HYBRIDIZE

amp

LABEL

gDNAPCR

SNP typing CF

CYP 450 RVP


CNV etc

RVP FAST GPP FAST

HYBRIDIZE

amp

LABEL

TAG1

TAG2 ANNEAL

amp

PCR

gDNA


30



2 hours



30 minutes



30 minutes





30 minutes







methods







TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

AAAAAAA RNA

AAAAAAA RNA

AAAAAAA RNA






TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

AAAAAAA RNA

AAAAAAA RNA

AAAAAAA RNA

cDNA

cDNA

cDNA




TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

cDNA

cDNA

cDNA




LMA Probe Design



Upstream LMA Probe

cDNA


Universal Primer 1

5rsquo 3rsquo












TAG

SAPE

Detection



lt3 Hours Setup











ANNEAL

Ligate

PCR

TAG1

TAG2

cDNA or

gDNAPCR

HYBRIDIZE

amp

LABEL





Upstream LMA Probes

T

Universal Primer 1

5rsquo

A

5rsquo

G



SNP

T



T

T


PCR Amplification

UP 1

P

No Amplification

B-UP 2



Universal Prime mix

UP 1

P

B-UP 2

UP 1

P

B-UP 2

Ligate

T T







TAG

SAPE

Detection



Bead Hyb 80 minutes







Incubation








Targeted PCR




Upstream LDR Probes

A T

G



SNP

A

A



A


Multiple

Ligation

cycles









TAG

SAPE

Detection






Forward primers

gDNA


5rsquo

3rsquo


A

T

5rsquo

3rsquo

G





Targeted PCR



A

5rsquo

3rsquo

G

5rsquo

3rsquo

T




A

5rsquo

3rsquo

G 5rsquo

3rsquo

T








xTAG


Detection

SAPE










xTAG primer

gDNA


Biotinylated primer

5rsquo 3rsquo

3rsquo

dATP dTTP dGTP dCTP

= Carbon Spacer











Detection

Sequence Detection

TAG


















Workflow

75



2 hours



30 minutes



30 minutes





30 minutes

76






0

500

1000

1500

2000

2500

3000

3500

4000


Sample

Sig

nal In

ten

sit

y (

MF

I)

let-7c

Let-7a

77


miR-107 vs miR-103

0

1000

2000

3000

4000

5000

6000


Sample

Sig

na

l In

ten

sit

y (

MF

I)

miR-103

miR-107









0

1000

2000

3000

4000

5000

6000

7000

8000

9000

10000

hsa-m

iR-1

07

hsa-m

iR-1

47

hsa-m

iR-1

81a

hsa-m

iR-1

92

hsa-m

iR-1

9b

hsa-m

iR-2

20a

hsa-m

iR-2

1hsa-m

iR-2

18

hsa-le

t-7c

hsa-m

iR-1

00

hsa-m

iR-1

25b

hsa-m

iR-

hsa-m

iR-2

05

hsa-m

iR-2

21

hsa-m

iR-2

00a

hsa-m

iR-2

00c

hsa-m

iR-2

06

hsa-m

iR-1

0a

hsa-m

iR-1

6

hsa-m

iR-9

hsa-m

iR-4

24

hsa-m

iR-2

0a

hsa-m

iR-2

0b

hsa-le

t-7a

hsa-m

iR-9

5hsa-m

iR-1

36

hsa-m

iR-1

95

hsa-m

iR-2

10

hsa-m

iR-2

12

hsa-m

iR-

hsa-m

iR-1

7

hsa-m

iR-1

22

hsa-m

iR-1

43

hsa-m

iR-1

45

hsa-m

iR-1

06a

hsa-m

iR-

hsa-m

iR-1

0b

hsa-m

iR-1

hsa-m

iR-2

23

hsa-m

iR-1

81b

hsa-m

iR-1

55

hsa-le

t-7g

hsa-le

t-7i

hsa-m

iR-9

6

hsa-m

iR-1

37

hsa-m

iR-1

85

hsa-m

iR-1

38

hsa-m

iR-1

98

hsa-m

iR-1

33a

hsa-m

iR-1

33b

hsa-m

iR-1

5a

hsa-m

iR-3

4a

hsa-m

iR-2

9a

hsa-m

iR-2

9b

hsa-m

iR-1

03

hsa-m

iR-2

22

hsa-m

iR-1

46a

cel-m

iR-3

9cel-m

iR-5

4

cel-m

iR-2

38

Regions

Ne

t S

ign

al In

ten

sit

y (

MF

I)

106a

17


Total RNA Samples

53-Plex Assay

miR-9 miR-122 miR-1

0

5000

10000

15000

20000

25000

Samples

Ne

t S

ign

al In

ten

sit

y (

MF

I)

miR-9

miR-122

miR-1






289704

12431797

4050

7044

12899

18030

24973

23884

21852

19249

11326

6573

3852

2471

0

5000

10000

15000

20000

25000

30000


Input Amount

Ne

t S

ign

al In

ten

sit

y (

MF

I)

200 amol


81


0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000



Ne

t S

ign

al in

ten

sit

y (

MF

I)

82

miR-133a

000

020

040

060

080

100

120


Total RNA Samples

Rela

tive C

han

ge t

o H

eart

FlexmiR v2

qRT-PCR

Tissue

Re

lati

ve

Ch

an

ge


LNP-miRNA

qRT-PCR

83

miRNA

Re

lati

ve

Ch

an

ge



0

5

10

15

20

25


miRNA

Rela

tive C

han

ge

FlexmiR v2

qRT-PCR

LNP-miRNA

qPCR

84



0

5000

10000

15000

20000

25000

30000

hsa-m

iR-1

07

hsa-m

iR-1

47

hsa-m

iR-1

81a

hsa-m

iR-1

92

hsa-m

iR-1

9b

hsa-m

iR-2

20a

hsa-m

iR-2

1hsa-m

iR-2

18

hsa-le

t-7c

hsa-m

iR-1

00

hsa-m

iR-1

25b

hsa-m

iR-

hsa-m

iR-2

05

hsa-m

iR-2

21

hsa-m

iR-2

00a

hsa-m

iR-2

00c

hsa-m

iR-2

06

hsa-m

iR-1

0a

hsa-m

iR-1

6hsa-m

iR-9

hsa-m

iR-4

24

hsa-m

iR-2

0a

hsa-m

iR-2

0b

hsa-le

t-7a

hsa-m

iR-9

5hsa-m

iR-1

36

hsa-m

iR-1

95

hsa-m

iR-2

10

hsa-m

iR-2

12

hsa-m

iR-

hsa-m

iR-1

7hsa-m

iR-1

22

hsa-m

iR-1

43

hsa-m

iR-1

45

hsa-m

ir-1

06a

hsa-m

iR-

hsa-m

iR-1

0b

hsa-m

iR-1

hsa-m

iR-2

23

hsa-m

iR-1

81b

hsa-le

t-155

hsa-le

t-7g

hsa-m

iR-7

ihsa-m

iR-9

6hsa-m

iR-1

37

hsa-m

iR-1

85

hsa-m

iR-1

38

hsa-m

iR-1

33a

hsa-m

iR-1

33b

hsa-m

iR-1

5a

hsa-m

iR-3

4a

hsa-m

iR-2

9a

hsa-m

iR-2

9b

hsa-m

iR-1

03

hsa-m

iR-2

22

hsa-m

iR-1

46a

hsa-m

iR-3

9cel-m

iR-3

9cel-m

iR-2

38

Regions

Ne

t S

ign

al in

ten

sit

y (

MF

I)

50ng

100ng

250ng

500ng








bull Flexible





Proteomic Assays



Nucleic Acids








or IgM





Multiplex Detection

Detection


IgG

1 2 3 4





















Y Y

Y

IgG1 IgG2

IgG3

IgG4 IgA



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Detection



Antigen

1 2 3








Y Y Y

Y

Y

Y Y

Y Y

Y Y

Y



Y


Detection

Multiplex Detection


















compounds





compounds



SAPE SAPE














SAPE SAPE


Detection

Multiplex Detection












543-550






GTPases 827 271-282














Single Plex Assays

To Luminex

Multiplex

Assays

















MagPlextrade








MicroPlextrade

Microspheres 56 um





















plate



plates




of a 96 well plate




well plates



Real Examples

Arrays

And

NGS

RT-PCR


Discovery

amp

Validation-1

An

aly

tes

S

am

ple

Samples Day

10s 100s 1000s 10000s

10

100

1000

10000 3D Throughput



6 x 384 platesday

2304 samplesday

1152000 dpday


MAGPIX

50 Plex

LX200

80-100 Plex

FLEXMAP 3D

500 Plex




concentrations

Quantify binding

events

Interrogate bead

with red laser

(635 nm)

Interrogate label

with green laser

(525 nm)

Sheath Fluid



green LED (525 nm)


LED (635 nm)


CCD imager


Capture


Genomic Assays










Detection

SAPE






HPV (high-risk

mucosal anogenital)




Quint et al 2009



HPV (high-risk

mucosal anogenital)



HPV (mucosal

anogenital)










Detection

RNA

Biotinylated cDNA








1 2





1

2

TAG-1

TAG-2



o Gene expression



o miRNA analysis


applications




ANNEAL

Ligate

PCR

TAG1

TAG2

cDNA or

gDNAPCR

HYBRIDIZE

amp

LABEL

dA

dG dT

dC -

ANNEAL

amp

Extend

TAG1

TAG2

HYBRIDIZE

amp

LABEL

gDNAPCR

SNP typing CF

CYP 450 RVP


CNV etc

RVP FAST GPP FAST

HYBRIDIZE

amp

LABEL

TAG1

TAG2 ANNEAL

amp

PCR

gDNA


30



2 hours



30 minutes



30 minutes





30 minutes







methods







TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

AAAAAAA RNA

AAAAAAA RNA

AAAAAAA RNA






TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

AAAAAAA RNA

AAAAAAA RNA

AAAAAAA RNA

cDNA

cDNA

cDNA




TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

cDNA

cDNA

cDNA




LMA Probe Design



Upstream LMA Probe

cDNA


Universal Primer 1

5rsquo 3rsquo












TAG

SAPE

Detection



lt3 Hours Setup











ANNEAL

Ligate

PCR

TAG1

TAG2

cDNA or

gDNAPCR

HYBRIDIZE

amp

LABEL





Upstream LMA Probes

T

Universal Primer 1

5rsquo

A

5rsquo

G



SNP

T



T

T


PCR Amplification

UP 1

P

No Amplification

B-UP 2



Universal Prime mix

UP 1

P

B-UP 2

UP 1

P

B-UP 2

Ligate

T T







TAG

SAPE

Detection



Bead Hyb 80 minutes







Incubation








Targeted PCR




Upstream LDR Probes

A T

G



SNP

A

A



A


Multiple

Ligation

cycles









TAG

SAPE

Detection






Forward primers

gDNA


5rsquo

3rsquo


A

T

5rsquo

3rsquo

G





Targeted PCR



A

5rsquo

3rsquo

G

5rsquo

3rsquo

T




A

5rsquo

3rsquo

G 5rsquo

3rsquo

T








xTAG


Detection

SAPE










xTAG primer

gDNA


Biotinylated primer

5rsquo 3rsquo

3rsquo

dATP dTTP dGTP dCTP

= Carbon Spacer











Detection

Sequence Detection

TAG


















Workflow

75



2 hours



30 minutes



30 minutes





30 minutes

76






0

500

1000

1500

2000

2500

3000

3500

4000


Sample

Sig

nal In

ten

sit

y (

MF

I)

let-7c

Let-7a

77


miR-107 vs miR-103

0

1000

2000

3000

4000

5000

6000


Sample

Sig

na

l In

ten

sit

y (

MF

I)

miR-103

miR-107









0

1000

2000

3000

4000

5000

6000

7000

8000

9000

10000

hsa-m

iR-1

07

hsa-m

iR-1

47

hsa-m

iR-1

81a

hsa-m

iR-1

92

hsa-m

iR-1

9b

hsa-m

iR-2

20a

hsa-m

iR-2

1hsa-m

iR-2

18

hsa-le

t-7c

hsa-m

iR-1

00

hsa-m

iR-1

25b

hsa-m

iR-

hsa-m

iR-2

05

hsa-m

iR-2

21

hsa-m

iR-2

00a

hsa-m

iR-2

00c

hsa-m

iR-2

06

hsa-m

iR-1

0a

hsa-m

iR-1

6

hsa-m

iR-9

hsa-m

iR-4

24

hsa-m

iR-2

0a

hsa-m

iR-2

0b

hsa-le

t-7a

hsa-m

iR-9

5hsa-m

iR-1

36

hsa-m

iR-1

95

hsa-m

iR-2

10

hsa-m

iR-2

12

hsa-m

iR-

hsa-m

iR-1

7

hsa-m

iR-1

22

hsa-m

iR-1

43

hsa-m

iR-1

45

hsa-m

iR-1

06a

hsa-m

iR-

hsa-m

iR-1

0b

hsa-m

iR-1

hsa-m

iR-2

23

hsa-m

iR-1

81b

hsa-m

iR-1

55

hsa-le

t-7g

hsa-le

t-7i

hsa-m

iR-9

6

hsa-m

iR-1

37

hsa-m

iR-1

85

hsa-m

iR-1

38

hsa-m

iR-1

98

hsa-m

iR-1

33a

hsa-m

iR-1

33b

hsa-m

iR-1

5a

hsa-m

iR-3

4a

hsa-m

iR-2

9a

hsa-m

iR-2

9b

hsa-m

iR-1

03

hsa-m

iR-2

22

hsa-m

iR-1

46a

cel-m

iR-3

9cel-m

iR-5

4

cel-m

iR-2

38

Regions

Ne

t S

ign

al In

ten

sit

y (

MF

I)

106a

17


Total RNA Samples

53-Plex Assay

miR-9 miR-122 miR-1

0

5000

10000

15000

20000

25000

Samples

Ne

t S

ign

al In

ten

sit

y (

MF

I)

miR-9

miR-122

miR-1






289704

12431797

4050

7044

12899

18030

24973

23884

21852

19249

11326

6573

3852

2471

0

5000

10000

15000

20000

25000

30000


Input Amount

Ne

t S

ign

al In

ten

sit

y (

MF

I)

200 amol


81


0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000



Ne

t S

ign

al in

ten

sit

y (

MF

I)

82

miR-133a

000

020

040

060

080

100

120


Total RNA Samples

Rela

tive C

han

ge t

o H

eart

FlexmiR v2

qRT-PCR

Tissue

Re

lati

ve

Ch

an

ge


LNP-miRNA

qRT-PCR

83

miRNA

Re

lati

ve

Ch

an

ge



0

5

10

15

20

25


miRNA

Rela

tive C

han

ge

FlexmiR v2

qRT-PCR

LNP-miRNA

qPCR

84



0

5000

10000

15000

20000

25000

30000

hsa-m

iR-1

07

hsa-m

iR-1

47

hsa-m

iR-1

81a

hsa-m

iR-1

92

hsa-m

iR-1

9b

hsa-m

iR-2

20a

hsa-m

iR-2

1hsa-m

iR-2

18

hsa-le

t-7c

hsa-m

iR-1

00

hsa-m

iR-1

25b

hsa-m

iR-

hsa-m

iR-2

05

hsa-m

iR-2

21

hsa-m

iR-2

00a

hsa-m

iR-2

00c

hsa-m

iR-2

06

hsa-m

iR-1

0a

hsa-m

iR-1

6hsa-m

iR-9

hsa-m

iR-4

24

hsa-m

iR-2

0a

hsa-m

iR-2

0b

hsa-le

t-7a

hsa-m

iR-9

5hsa-m

iR-1

36

hsa-m

iR-1

95

hsa-m

iR-2

10

hsa-m

iR-2

12

hsa-m

iR-

hsa-m

iR-1

7hsa-m

iR-1

22

hsa-m

iR-1

43

hsa-m

iR-1

45

hsa-m

ir-1

06a

hsa-m

iR-

hsa-m

iR-1

0b

hsa-m

iR-1

hsa-m

iR-2

23

hsa-m

iR-1

81b

hsa-le

t-155

hsa-le

t-7g

hsa-m

iR-7

ihsa-m

iR-9

6hsa-m

iR-1

37

hsa-m

iR-1

85

hsa-m

iR-1

38

hsa-m

iR-1

33a

hsa-m

iR-1

33b

hsa-m

iR-1

5a

hsa-m

iR-3

4a

hsa-m

iR-2

9a

hsa-m

iR-2

9b

hsa-m

iR-1

03

hsa-m

iR-2

22

hsa-m

iR-1

46a

hsa-m

iR-3

9cel-m

iR-3

9cel-m

iR-2

38

Regions

Ne

t S

ign

al in

ten

sit

y (

MF

I)

50ng

100ng

250ng

500ng








bull Flexible





Proteomic Assays



Nucleic Acids








or IgM





Multiplex Detection

Detection


IgG

1 2 3 4





















Y Y

Y

IgG1 IgG2

IgG3

IgG4 IgA



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Detection



Antigen

1 2 3








Y Y Y

Y

Y

Y Y

Y Y

Y Y

Y



Y


Detection

Multiplex Detection


















compounds





compounds



SAPE SAPE














SAPE SAPE


Detection

Multiplex Detection












543-550






GTPases 827 271-282














Single Plex Assays

To Luminex

Multiplex

Assays















MagPlextrade








MicroPlextrade

Microspheres 56 um





















plate



plates




of a 96 well plate




well plates



Real Examples

Arrays

And

NGS

RT-PCR


Discovery

amp

Validation-1

An

aly

tes

S

am

ple

Samples Day

10s 100s 1000s 10000s

10

100

1000

10000 3D Throughput



6 x 384 platesday

2304 samplesday

1152000 dpday


MAGPIX

50 Plex

LX200

80-100 Plex

FLEXMAP 3D

500 Plex




concentrations

Quantify binding

events

Interrogate bead

with red laser

(635 nm)

Interrogate label

with green laser

(525 nm)

Sheath Fluid



green LED (525 nm)


LED (635 nm)


CCD imager


Capture


Genomic Assays










Detection

SAPE






HPV (high-risk

mucosal anogenital)




Quint et al 2009



HPV (high-risk

mucosal anogenital)



HPV (mucosal

anogenital)










Detection

RNA

Biotinylated cDNA








1 2





1

2

TAG-1

TAG-2



o Gene expression



o miRNA analysis


applications




ANNEAL

Ligate

PCR

TAG1

TAG2

cDNA or

gDNAPCR

HYBRIDIZE

amp

LABEL

dA

dG dT

dC -

ANNEAL

amp

Extend

TAG1

TAG2

HYBRIDIZE

amp

LABEL

gDNAPCR

SNP typing CF

CYP 450 RVP


CNV etc

RVP FAST GPP FAST

HYBRIDIZE

amp

LABEL

TAG1

TAG2 ANNEAL

amp

PCR

gDNA


30



2 hours



30 minutes



30 minutes





30 minutes







methods







TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

AAAAAAA RNA

AAAAAAA RNA

AAAAAAA RNA






TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

AAAAAAA RNA

AAAAAAA RNA

AAAAAAA RNA

cDNA

cDNA

cDNA




TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

cDNA

cDNA

cDNA




LMA Probe Design



Upstream LMA Probe

cDNA


Universal Primer 1

5rsquo 3rsquo












TAG

SAPE

Detection



lt3 Hours Setup











ANNEAL

Ligate

PCR

TAG1

TAG2

cDNA or

gDNAPCR

HYBRIDIZE

amp

LABEL





Upstream LMA Probes

T

Universal Primer 1

5rsquo

A

5rsquo

G



SNP

T



T

T


PCR Amplification

UP 1

P

No Amplification

B-UP 2



Universal Prime mix

UP 1

P

B-UP 2

UP 1

P

B-UP 2

Ligate

T T







TAG

SAPE

Detection



Bead Hyb 80 minutes







Incubation








Targeted PCR




Upstream LDR Probes

A T

G



SNP

A

A



A


Multiple

Ligation

cycles









TAG

SAPE

Detection






Forward primers

gDNA


5rsquo

3rsquo


A

T

5rsquo

3rsquo

G





Targeted PCR



A

5rsquo

3rsquo

G

5rsquo

3rsquo

T




A

5rsquo

3rsquo

G 5rsquo

3rsquo

T








xTAG


Detection

SAPE










xTAG primer

gDNA


Biotinylated primer

5rsquo 3rsquo

3rsquo

dATP dTTP dGTP dCTP

= Carbon Spacer











Detection

Sequence Detection

TAG


















Workflow

75



2 hours



30 minutes



30 minutes





30 minutes

76






0

500

1000

1500

2000

2500

3000

3500

4000


Sample

Sig

nal In

ten

sit

y (

MF

I)

let-7c

Let-7a

77


miR-107 vs miR-103

0

1000

2000

3000

4000

5000

6000


Sample

Sig

na

l In

ten

sit

y (

MF

I)

miR-103

miR-107









0

1000

2000

3000

4000

5000

6000

7000

8000

9000

10000

hsa-m

iR-1

07

hsa-m

iR-1

47

hsa-m

iR-1

81a

hsa-m

iR-1

92

hsa-m

iR-1

9b

hsa-m

iR-2

20a

hsa-m

iR-2

1hsa-m

iR-2

18

hsa-le

t-7c

hsa-m

iR-1

00

hsa-m

iR-1

25b

hsa-m

iR-

hsa-m

iR-2

05

hsa-m

iR-2

21

hsa-m

iR-2

00a

hsa-m

iR-2

00c

hsa-m

iR-2

06

hsa-m

iR-1

0a

hsa-m

iR-1

6

hsa-m

iR-9

hsa-m

iR-4

24

hsa-m

iR-2

0a

hsa-m

iR-2

0b

hsa-le

t-7a

hsa-m

iR-9

5hsa-m

iR-1

36

hsa-m

iR-1

95

hsa-m

iR-2

10

hsa-m

iR-2

12

hsa-m

iR-

hsa-m

iR-1

7

hsa-m

iR-1

22

hsa-m

iR-1

43

hsa-m

iR-1

45

hsa-m

iR-1

06a

hsa-m

iR-

hsa-m

iR-1

0b

hsa-m

iR-1

hsa-m

iR-2

23

hsa-m

iR-1

81b

hsa-m

iR-1

55

hsa-le

t-7g

hsa-le

t-7i

hsa-m

iR-9

6

hsa-m

iR-1

37

hsa-m

iR-1

85

hsa-m

iR-1

38

hsa-m

iR-1

98

hsa-m

iR-1

33a

hsa-m

iR-1

33b

hsa-m

iR-1

5a

hsa-m

iR-3

4a

hsa-m

iR-2

9a

hsa-m

iR-2

9b

hsa-m

iR-1

03

hsa-m

iR-2

22

hsa-m

iR-1

46a

cel-m

iR-3

9cel-m

iR-5

4

cel-m

iR-2

38

Regions

Ne

t S

ign

al In

ten

sit

y (

MF

I)

106a

17


Total RNA Samples

53-Plex Assay

miR-9 miR-122 miR-1

0

5000

10000

15000

20000

25000

Samples

Ne

t S

ign

al In

ten

sit

y (

MF

I)

miR-9

miR-122

miR-1






289704

12431797

4050

7044

12899

18030

24973

23884

21852

19249

11326

6573

3852

2471

0

5000

10000

15000

20000

25000

30000


Input Amount

Ne

t S

ign

al In

ten

sit

y (

MF

I)

200 amol


81


0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000



Ne

t S

ign

al in

ten

sit

y (

MF

I)

82

miR-133a

000

020

040

060

080

100

120


Total RNA Samples

Rela

tive C

han

ge t

o H

eart

FlexmiR v2

qRT-PCR

Tissue

Re

lati

ve

Ch

an

ge


LNP-miRNA

qRT-PCR

83

miRNA

Re

lati

ve

Ch

an

ge



0

5

10

15

20

25


miRNA

Rela

tive C

han

ge

FlexmiR v2

qRT-PCR

LNP-miRNA

qPCR

84



0

5000

10000

15000

20000

25000

30000

hsa-m

iR-1

07

hsa-m

iR-1

47

hsa-m

iR-1

81a

hsa-m

iR-1

92

hsa-m

iR-1

9b

hsa-m

iR-2

20a

hsa-m

iR-2

1hsa-m

iR-2

18

hsa-le

t-7c

hsa-m

iR-1

00

hsa-m

iR-1

25b

hsa-m

iR-

hsa-m

iR-2

05

hsa-m

iR-2

21

hsa-m

iR-2

00a

hsa-m

iR-2

00c

hsa-m

iR-2

06

hsa-m

iR-1

0a

hsa-m

iR-1

6hsa-m

iR-9

hsa-m

iR-4

24

hsa-m

iR-2

0a

hsa-m

iR-2

0b

hsa-le

t-7a

hsa-m

iR-9

5hsa-m

iR-1

36

hsa-m

iR-1

95

hsa-m

iR-2

10

hsa-m

iR-2

12

hsa-m

iR-

hsa-m

iR-1

7hsa-m

iR-1

22

hsa-m

iR-1

43

hsa-m

iR-1

45

hsa-m

ir-1

06a

hsa-m

iR-

hsa-m

iR-1

0b

hsa-m

iR-1

hsa-m

iR-2

23

hsa-m

iR-1

81b

hsa-le

t-155

hsa-le

t-7g

hsa-m

iR-7

ihsa-m

iR-9

6hsa-m

iR-1

37

hsa-m

iR-1

85

hsa-m

iR-1

38

hsa-m

iR-1

33a

hsa-m

iR-1

33b

hsa-m

iR-1

5a

hsa-m

iR-3

4a

hsa-m

iR-2

9a

hsa-m

iR-2

9b

hsa-m

iR-1

03

hsa-m

iR-2

22

hsa-m

iR-1

46a

hsa-m

iR-3

9cel-m

iR-3

9cel-m

iR-2

38

Regions

Ne

t S

ign

al in

ten

sit

y (

MF

I)

50ng

100ng

250ng

500ng








bull Flexible





Proteomic Assays



Nucleic Acids








or IgM





Multiplex Detection

Detection


IgG

1 2 3 4





















Y Y

Y

IgG1 IgG2

IgG3

IgG4 IgA



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Detection



Antigen

1 2 3








Y Y Y

Y

Y

Y Y

Y Y

Y Y

Y



Y


Detection

Multiplex Detection


















compounds





compounds



SAPE SAPE














SAPE SAPE


Detection

Multiplex Detection












543-550






GTPases 827 271-282














Single Plex Assays

To Luminex

Multiplex

Assays













MagPlextrade








MicroPlextrade

Microspheres 56 um





















plate



plates




of a 96 well plate




well plates



Real Examples

Arrays

And

NGS

RT-PCR


Discovery

amp

Validation-1

An

aly

tes

S

am

ple

Samples Day

10s 100s 1000s 10000s

10

100

1000

10000 3D Throughput



6 x 384 platesday

2304 samplesday

1152000 dpday


MAGPIX

50 Plex

LX200

80-100 Plex

FLEXMAP 3D

500 Plex




concentrations

Quantify binding

events

Interrogate bead

with red laser

(635 nm)

Interrogate label

with green laser

(525 nm)

Sheath Fluid



green LED (525 nm)


LED (635 nm)


CCD imager


Capture


Genomic Assays










Detection

SAPE






HPV (high-risk

mucosal anogenital)




Quint et al 2009



HPV (high-risk

mucosal anogenital)



HPV (mucosal

anogenital)










Detection

RNA

Biotinylated cDNA








1 2





1

2

TAG-1

TAG-2



o Gene expression



o miRNA analysis


applications




ANNEAL

Ligate

PCR

TAG1

TAG2

cDNA or

gDNAPCR

HYBRIDIZE

amp

LABEL

dA

dG dT

dC -

ANNEAL

amp

Extend

TAG1

TAG2

HYBRIDIZE

amp

LABEL

gDNAPCR

SNP typing CF

CYP 450 RVP


CNV etc

RVP FAST GPP FAST

HYBRIDIZE

amp

LABEL

TAG1

TAG2 ANNEAL

amp

PCR

gDNA


30



2 hours



30 minutes



30 minutes





30 minutes







methods







TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

AAAAAAA RNA

AAAAAAA RNA

AAAAAAA RNA






TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

AAAAAAA RNA

AAAAAAA RNA

AAAAAAA RNA

cDNA

cDNA

cDNA




TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

cDNA

cDNA

cDNA




LMA Probe Design



Upstream LMA Probe

cDNA


Universal Primer 1

5rsquo 3rsquo












TAG

SAPE

Detection



lt3 Hours Setup











ANNEAL

Ligate

PCR

TAG1

TAG2

cDNA or

gDNAPCR

HYBRIDIZE

amp

LABEL





Upstream LMA Probes

T

Universal Primer 1

5rsquo

A

5rsquo

G



SNP

T



T

T


PCR Amplification

UP 1

P

No Amplification

B-UP 2



Universal Prime mix

UP 1

P

B-UP 2

UP 1

P

B-UP 2

Ligate

T T







TAG

SAPE

Detection



Bead Hyb 80 minutes







Incubation








Targeted PCR




Upstream LDR Probes

A T

G



SNP

A

A



A


Multiple

Ligation

cycles









TAG

SAPE

Detection






Forward primers

gDNA


5rsquo

3rsquo


A

T

5rsquo

3rsquo

G





Targeted PCR



A

5rsquo

3rsquo

G

5rsquo

3rsquo

T




A

5rsquo

3rsquo

G 5rsquo

3rsquo

T








xTAG


Detection

SAPE










xTAG primer

gDNA


Biotinylated primer

5rsquo 3rsquo

3rsquo

dATP dTTP dGTP dCTP

= Carbon Spacer











Detection

Sequence Detection

TAG


















Workflow

75



2 hours



30 minutes



30 minutes





30 minutes

76






0

500

1000

1500

2000

2500

3000

3500

4000


Sample

Sig

nal In

ten

sit

y (

MF

I)

let-7c

Let-7a

77


miR-107 vs miR-103

0

1000

2000

3000

4000

5000

6000


Sample

Sig

na

l In

ten

sit

y (

MF

I)

miR-103

miR-107









0

1000

2000

3000

4000

5000

6000

7000

8000

9000

10000

hsa-m

iR-1

07

hsa-m

iR-1

47

hsa-m

iR-1

81a

hsa-m

iR-1

92

hsa-m

iR-1

9b

hsa-m

iR-2

20a

hsa-m

iR-2

1hsa-m

iR-2

18

hsa-le

t-7c

hsa-m

iR-1

00

hsa-m

iR-1

25b

hsa-m

iR-

hsa-m

iR-2

05

hsa-m

iR-2

21

hsa-m

iR-2

00a

hsa-m

iR-2

00c

hsa-m

iR-2

06

hsa-m

iR-1

0a

hsa-m

iR-1

6

hsa-m

iR-9

hsa-m

iR-4

24

hsa-m

iR-2

0a

hsa-m

iR-2

0b

hsa-le

t-7a

hsa-m

iR-9

5hsa-m

iR-1

36

hsa-m

iR-1

95

hsa-m

iR-2

10

hsa-m

iR-2

12

hsa-m

iR-

hsa-m

iR-1

7

hsa-m

iR-1

22

hsa-m

iR-1

43

hsa-m

iR-1

45

hsa-m

iR-1

06a

hsa-m

iR-

hsa-m

iR-1

0b

hsa-m

iR-1

hsa-m

iR-2

23

hsa-m

iR-1

81b

hsa-m

iR-1

55

hsa-le

t-7g

hsa-le

t-7i

hsa-m

iR-9

6

hsa-m

iR-1

37

hsa-m

iR-1

85

hsa-m

iR-1

38

hsa-m

iR-1

98

hsa-m

iR-1

33a

hsa-m

iR-1

33b

hsa-m

iR-1

5a

hsa-m

iR-3

4a

hsa-m

iR-2

9a

hsa-m

iR-2

9b

hsa-m

iR-1

03

hsa-m

iR-2

22

hsa-m

iR-1

46a

cel-m

iR-3

9cel-m

iR-5

4

cel-m

iR-2

38

Regions

Ne

t S

ign

al In

ten

sit

y (

MF

I)

106a

17


Total RNA Samples

53-Plex Assay

miR-9 miR-122 miR-1

0

5000

10000

15000

20000

25000

Samples

Ne

t S

ign

al In

ten

sit

y (

MF

I)

miR-9

miR-122

miR-1






289704

12431797

4050

7044

12899

18030

24973

23884

21852

19249

11326

6573

3852

2471

0

5000

10000

15000

20000

25000

30000


Input Amount

Ne

t S

ign

al In

ten

sit

y (

MF

I)

200 amol


81


0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000



Ne

t S

ign

al in

ten

sit

y (

MF

I)

82

miR-133a

000

020

040

060

080

100

120


Total RNA Samples

Rela

tive C

han

ge t

o H

eart

FlexmiR v2

qRT-PCR

Tissue

Re

lati

ve

Ch

an

ge


LNP-miRNA

qRT-PCR

83

miRNA

Re

lati

ve

Ch

an

ge



0

5

10

15

20

25


miRNA

Rela

tive C

han

ge

FlexmiR v2

qRT-PCR

LNP-miRNA

qPCR

84



0

5000

10000

15000

20000

25000

30000

hsa-m

iR-1

07

hsa-m

iR-1

47

hsa-m

iR-1

81a

hsa-m

iR-1

92

hsa-m

iR-1

9b

hsa-m

iR-2

20a

hsa-m

iR-2

1hsa-m

iR-2

18

hsa-le

t-7c

hsa-m

iR-1

00

hsa-m

iR-1

25b

hsa-m

iR-

hsa-m

iR-2

05

hsa-m

iR-2

21

hsa-m

iR-2

00a

hsa-m

iR-2

00c

hsa-m

iR-2

06

hsa-m

iR-1

0a

hsa-m

iR-1

6hsa-m

iR-9

hsa-m

iR-4

24

hsa-m

iR-2

0a

hsa-m

iR-2

0b

hsa-le

t-7a

hsa-m

iR-9

5hsa-m

iR-1

36

hsa-m

iR-1

95

hsa-m

iR-2

10

hsa-m

iR-2

12

hsa-m

iR-

hsa-m

iR-1

7hsa-m

iR-1

22

hsa-m

iR-1

43

hsa-m

iR-1

45

hsa-m

ir-1

06a

hsa-m

iR-

hsa-m

iR-1

0b

hsa-m

iR-1

hsa-m

iR-2

23

hsa-m

iR-1

81b

hsa-le

t-155

hsa-le

t-7g

hsa-m

iR-7

ihsa-m

iR-9

6hsa-m

iR-1

37

hsa-m

iR-1

85

hsa-m

iR-1

38

hsa-m

iR-1

33a

hsa-m

iR-1

33b

hsa-m

iR-1

5a

hsa-m

iR-3

4a

hsa-m

iR-2

9a

hsa-m

iR-2

9b

hsa-m

iR-1

03

hsa-m

iR-2

22

hsa-m

iR-1

46a

hsa-m

iR-3

9cel-m

iR-3

9cel-m

iR-2

38

Regions

Ne

t S

ign

al in

ten

sit

y (

MF

I)

50ng

100ng

250ng

500ng








bull Flexible





Proteomic Assays



Nucleic Acids








or IgM





Multiplex Detection

Detection


IgG

1 2 3 4





















Y Y

Y

IgG1 IgG2

IgG3

IgG4 IgA



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Detection



Antigen

1 2 3








Y Y Y

Y

Y

Y Y

Y Y

Y Y

Y



Y


Detection

Multiplex Detection


















compounds





compounds



SAPE SAPE














SAPE SAPE


Detection

Multiplex Detection












543-550






GTPases 827 271-282














Single Plex Assays

To Luminex

Multiplex

Assays










MagPlextrade








MicroPlextrade

Microspheres 56 um





















plate



plates




of a 96 well plate




well plates



Real Examples

Arrays

And

NGS

RT-PCR


Discovery

amp

Validation-1

An

aly

tes

S

am

ple

Samples Day

10s 100s 1000s 10000s

10

100

1000

10000 3D Throughput



6 x 384 platesday

2304 samplesday

1152000 dpday


MAGPIX

50 Plex

LX200

80-100 Plex

FLEXMAP 3D

500 Plex




concentrations

Quantify binding

events

Interrogate bead

with red laser

(635 nm)

Interrogate label

with green laser

(525 nm)

Sheath Fluid



green LED (525 nm)


LED (635 nm)


CCD imager


Capture


Genomic Assays










Detection

SAPE






HPV (high-risk

mucosal anogenital)




Quint et al 2009



HPV (high-risk

mucosal anogenital)



HPV (mucosal

anogenital)










Detection

RNA

Biotinylated cDNA








1 2





1

2

TAG-1

TAG-2



o Gene expression



o miRNA analysis


applications




ANNEAL

Ligate

PCR

TAG1

TAG2

cDNA or

gDNAPCR

HYBRIDIZE

amp

LABEL

dA

dG dT

dC -

ANNEAL

amp

Extend

TAG1

TAG2

HYBRIDIZE

amp

LABEL

gDNAPCR

SNP typing CF

CYP 450 RVP


CNV etc

RVP FAST GPP FAST

HYBRIDIZE

amp

LABEL

TAG1

TAG2 ANNEAL

amp

PCR

gDNA


30



2 hours



30 minutes



30 minutes





30 minutes







methods







TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

AAAAAAA RNA

AAAAAAA RNA

AAAAAAA RNA






TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

AAAAAAA RNA

AAAAAAA RNA

AAAAAAA RNA

cDNA

cDNA

cDNA




TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

cDNA

cDNA

cDNA




LMA Probe Design



Upstream LMA Probe

cDNA


Universal Primer 1

5rsquo 3rsquo












TAG

SAPE

Detection



lt3 Hours Setup











ANNEAL

Ligate

PCR

TAG1

TAG2

cDNA or

gDNAPCR

HYBRIDIZE

amp

LABEL





Upstream LMA Probes

T

Universal Primer 1

5rsquo

A

5rsquo

G



SNP

T



T

T


PCR Amplification

UP 1

P

No Amplification

B-UP 2



Universal Prime mix

UP 1

P

B-UP 2

UP 1

P

B-UP 2

Ligate

T T







TAG

SAPE

Detection



Bead Hyb 80 minutes







Incubation








Targeted PCR




Upstream LDR Probes

A T

G



SNP

A

A



A


Multiple

Ligation

cycles









TAG

SAPE

Detection






Forward primers

gDNA


5rsquo

3rsquo


A

T

5rsquo

3rsquo

G





Targeted PCR



A

5rsquo

3rsquo

G

5rsquo

3rsquo

T




A

5rsquo

3rsquo

G 5rsquo

3rsquo

T








xTAG


Detection

SAPE










xTAG primer

gDNA


Biotinylated primer

5rsquo 3rsquo

3rsquo

dATP dTTP dGTP dCTP

= Carbon Spacer











Detection

Sequence Detection

TAG


















Workflow

75



2 hours



30 minutes



30 minutes





30 minutes

76






0

500

1000

1500

2000

2500

3000

3500

4000


Sample

Sig

nal In

ten

sit

y (

MF

I)

let-7c

Let-7a

77


miR-107 vs miR-103

0

1000

2000

3000

4000

5000

6000


Sample

Sig

na

l In

ten

sit

y (

MF

I)

miR-103

miR-107









0

1000

2000

3000

4000

5000

6000

7000

8000

9000

10000

hsa-m

iR-1

07

hsa-m

iR-1

47

hsa-m

iR-1

81a

hsa-m

iR-1

92

hsa-m

iR-1

9b

hsa-m

iR-2

20a

hsa-m

iR-2

1hsa-m

iR-2

18

hsa-le

t-7c

hsa-m

iR-1

00

hsa-m

iR-1

25b

hsa-m

iR-

hsa-m

iR-2

05

hsa-m

iR-2

21

hsa-m

iR-2

00a

hsa-m

iR-2

00c

hsa-m

iR-2

06

hsa-m

iR-1

0a

hsa-m

iR-1

6

hsa-m

iR-9

hsa-m

iR-4

24

hsa-m

iR-2

0a

hsa-m

iR-2

0b

hsa-le

t-7a

hsa-m

iR-9

5hsa-m

iR-1

36

hsa-m

iR-1

95

hsa-m

iR-2

10

hsa-m

iR-2

12

hsa-m

iR-

hsa-m

iR-1

7

hsa-m

iR-1

22

hsa-m

iR-1

43

hsa-m

iR-1

45

hsa-m

iR-1

06a

hsa-m

iR-

hsa-m

iR-1

0b

hsa-m

iR-1

hsa-m

iR-2

23

hsa-m

iR-1

81b

hsa-m

iR-1

55

hsa-le

t-7g

hsa-le

t-7i

hsa-m

iR-9

6

hsa-m

iR-1

37

hsa-m

iR-1

85

hsa-m

iR-1

38

hsa-m

iR-1

98

hsa-m

iR-1

33a

hsa-m

iR-1

33b

hsa-m

iR-1

5a

hsa-m

iR-3

4a

hsa-m

iR-2

9a

hsa-m

iR-2

9b

hsa-m

iR-1

03

hsa-m

iR-2

22

hsa-m

iR-1

46a

cel-m

iR-3

9cel-m

iR-5

4

cel-m

iR-2

38

Regions

Ne

t S

ign

al In

ten

sit

y (

MF

I)

106a

17


Total RNA Samples

53-Plex Assay

miR-9 miR-122 miR-1

0

5000

10000

15000

20000

25000

Samples

Ne

t S

ign

al In

ten

sit

y (

MF

I)

miR-9

miR-122

miR-1






289704

12431797

4050

7044

12899

18030

24973

23884

21852

19249

11326

6573

3852

2471

0

5000

10000

15000

20000

25000

30000


Input Amount

Ne

t S

ign

al In

ten

sit

y (

MF

I)

200 amol


81


0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000



Ne

t S

ign

al in

ten

sit

y (

MF

I)

82

miR-133a

000

020

040

060

080

100

120


Total RNA Samples

Rela

tive C

han

ge t

o H

eart

FlexmiR v2

qRT-PCR

Tissue

Re

lati

ve

Ch

an

ge


LNP-miRNA

qRT-PCR

83

miRNA

Re

lati

ve

Ch

an

ge



0

5

10

15

20

25


miRNA

Rela

tive C

han

ge

FlexmiR v2

qRT-PCR

LNP-miRNA

qPCR

84



0

5000

10000

15000

20000

25000

30000

hsa-m

iR-1

07

hsa-m

iR-1

47

hsa-m

iR-1

81a

hsa-m

iR-1

92

hsa-m

iR-1

9b

hsa-m

iR-2

20a

hsa-m

iR-2

1hsa-m

iR-2

18

hsa-le

t-7c

hsa-m

iR-1

00

hsa-m

iR-1

25b

hsa-m

iR-

hsa-m

iR-2

05

hsa-m

iR-2

21

hsa-m

iR-2

00a

hsa-m

iR-2

00c

hsa-m

iR-2

06

hsa-m

iR-1

0a

hsa-m

iR-1

6hsa-m

iR-9

hsa-m

iR-4

24

hsa-m

iR-2

0a

hsa-m

iR-2

0b

hsa-le

t-7a

hsa-m

iR-9

5hsa-m

iR-1

36

hsa-m

iR-1

95

hsa-m

iR-2

10

hsa-m

iR-2

12

hsa-m

iR-

hsa-m

iR-1

7hsa-m

iR-1

22

hsa-m

iR-1

43

hsa-m

iR-1

45

hsa-m

ir-1

06a

hsa-m

iR-

hsa-m

iR-1

0b

hsa-m

iR-1

hsa-m

iR-2

23

hsa-m

iR-1

81b

hsa-le

t-155

hsa-le

t-7g

hsa-m

iR-7

ihsa-m

iR-9

6hsa-m

iR-1

37

hsa-m

iR-1

85

hsa-m

iR-1

38

hsa-m

iR-1

33a

hsa-m

iR-1

33b

hsa-m

iR-1

5a

hsa-m

iR-3

4a

hsa-m

iR-2

9a

hsa-m

iR-2

9b

hsa-m

iR-1

03

hsa-m

iR-2

22

hsa-m

iR-1

46a

hsa-m

iR-3

9cel-m

iR-3

9cel-m

iR-2

38

Regions

Ne

t S

ign

al in

ten

sit

y (

MF

I)

50ng

100ng

250ng

500ng








bull Flexible





Proteomic Assays



Nucleic Acids








or IgM





Multiplex Detection

Detection


IgG

1 2 3 4





















Y Y

Y

IgG1 IgG2

IgG3

IgG4 IgA



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Detection



Antigen

1 2 3








Y Y Y

Y

Y

Y Y

Y Y

Y Y

Y



Y


Detection

Multiplex Detection


















compounds





compounds



SAPE SAPE














SAPE SAPE


Detection

Multiplex Detection












543-550






GTPases 827 271-282














Single Plex Assays

To Luminex

Multiplex

Assays








MagPlextrade








MicroPlextrade

Microspheres 56 um





















plate



plates




of a 96 well plate




well plates



Real Examples

Arrays

And

NGS

RT-PCR


Discovery

amp

Validation-1

An

aly

tes

S

am

ple

Samples Day

10s 100s 1000s 10000s

10

100

1000

10000 3D Throughput



6 x 384 platesday

2304 samplesday

1152000 dpday


MAGPIX

50 Plex

LX200

80-100 Plex

FLEXMAP 3D

500 Plex




concentrations

Quantify binding

events

Interrogate bead

with red laser

(635 nm)

Interrogate label

with green laser

(525 nm)

Sheath Fluid



green LED (525 nm)


LED (635 nm)


CCD imager


Capture


Genomic Assays










Detection

SAPE






HPV (high-risk

mucosal anogenital)




Quint et al 2009



HPV (high-risk

mucosal anogenital)



HPV (mucosal

anogenital)










Detection

RNA

Biotinylated cDNA








1 2





1

2

TAG-1

TAG-2



o Gene expression



o miRNA analysis


applications




ANNEAL

Ligate

PCR

TAG1

TAG2

cDNA or

gDNAPCR

HYBRIDIZE

amp

LABEL

dA

dG dT

dC -

ANNEAL

amp

Extend

TAG1

TAG2

HYBRIDIZE

amp

LABEL

gDNAPCR

SNP typing CF

CYP 450 RVP


CNV etc

RVP FAST GPP FAST

HYBRIDIZE

amp

LABEL

TAG1

TAG2 ANNEAL

amp

PCR

gDNA


30



2 hours



30 minutes



30 minutes





30 minutes







methods







TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

AAAAAAA RNA

AAAAAAA RNA

AAAAAAA RNA






TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

AAAAAAA RNA

AAAAAAA RNA

AAAAAAA RNA

cDNA

cDNA

cDNA




TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

cDNA

cDNA

cDNA




LMA Probe Design



Upstream LMA Probe

cDNA


Universal Primer 1

5rsquo 3rsquo












TAG

SAPE

Detection



lt3 Hours Setup











ANNEAL

Ligate

PCR

TAG1

TAG2

cDNA or

gDNAPCR

HYBRIDIZE

amp

LABEL





Upstream LMA Probes

T

Universal Primer 1

5rsquo

A

5rsquo

G



SNP

T



T

T


PCR Amplification

UP 1

P

No Amplification

B-UP 2



Universal Prime mix

UP 1

P

B-UP 2

UP 1

P

B-UP 2

Ligate

T T







TAG

SAPE

Detection



Bead Hyb 80 minutes







Incubation








Targeted PCR




Upstream LDR Probes

A T

G



SNP

A

A



A


Multiple

Ligation

cycles









TAG

SAPE

Detection






Forward primers

gDNA


5rsquo

3rsquo


A

T

5rsquo

3rsquo

G





Targeted PCR



A

5rsquo

3rsquo

G

5rsquo

3rsquo

T




A

5rsquo

3rsquo

G 5rsquo

3rsquo

T








xTAG


Detection

SAPE










xTAG primer

gDNA


Biotinylated primer

5rsquo 3rsquo

3rsquo

dATP dTTP dGTP dCTP

= Carbon Spacer











Detection

Sequence Detection

TAG


















Workflow

75



2 hours



30 minutes



30 minutes





30 minutes

76






0

500

1000

1500

2000

2500

3000

3500

4000


Sample

Sig

nal In

ten

sit

y (

MF

I)

let-7c

Let-7a

77


miR-107 vs miR-103

0

1000

2000

3000

4000

5000

6000


Sample

Sig

na

l In

ten

sit

y (

MF

I)

miR-103

miR-107









0

1000

2000

3000

4000

5000

6000

7000

8000

9000

10000

hsa-m

iR-1

07

hsa-m

iR-1

47

hsa-m

iR-1

81a

hsa-m

iR-1

92

hsa-m

iR-1

9b

hsa-m

iR-2

20a

hsa-m

iR-2

1hsa-m

iR-2

18

hsa-le

t-7c

hsa-m

iR-1

00

hsa-m

iR-1

25b

hsa-m

iR-

hsa-m

iR-2

05

hsa-m

iR-2

21

hsa-m

iR-2

00a

hsa-m

iR-2

00c

hsa-m

iR-2

06

hsa-m

iR-1

0a

hsa-m

iR-1

6

hsa-m

iR-9

hsa-m

iR-4

24

hsa-m

iR-2

0a

hsa-m

iR-2

0b

hsa-le

t-7a

hsa-m

iR-9

5hsa-m

iR-1

36

hsa-m

iR-1

95

hsa-m

iR-2

10

hsa-m

iR-2

12

hsa-m

iR-

hsa-m

iR-1

7

hsa-m

iR-1

22

hsa-m

iR-1

43

hsa-m

iR-1

45

hsa-m

iR-1

06a

hsa-m

iR-

hsa-m

iR-1

0b

hsa-m

iR-1

hsa-m

iR-2

23

hsa-m

iR-1

81b

hsa-m

iR-1

55

hsa-le

t-7g

hsa-le

t-7i

hsa-m

iR-9

6

hsa-m

iR-1

37

hsa-m

iR-1

85

hsa-m

iR-1

38

hsa-m

iR-1

98

hsa-m

iR-1

33a

hsa-m

iR-1

33b

hsa-m

iR-1

5a

hsa-m

iR-3

4a

hsa-m

iR-2

9a

hsa-m

iR-2

9b

hsa-m

iR-1

03

hsa-m

iR-2

22

hsa-m

iR-1

46a

cel-m

iR-3

9cel-m

iR-5

4

cel-m

iR-2

38

Regions

Ne

t S

ign

al In

ten

sit

y (

MF

I)

106a

17


Total RNA Samples

53-Plex Assay

miR-9 miR-122 miR-1

0

5000

10000

15000

20000

25000

Samples

Ne

t S

ign

al In

ten

sit

y (

MF

I)

miR-9

miR-122

miR-1






289704

12431797

4050

7044

12899

18030

24973

23884

21852

19249

11326

6573

3852

2471

0

5000

10000

15000

20000

25000

30000


Input Amount

Ne

t S

ign

al In

ten

sit

y (

MF

I)

200 amol


81


0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000



Ne

t S

ign

al in

ten

sit

y (

MF

I)

82

miR-133a

000

020

040

060

080

100

120


Total RNA Samples

Rela

tive C

han

ge t

o H

eart

FlexmiR v2

qRT-PCR

Tissue

Re

lati

ve

Ch

an

ge


LNP-miRNA

qRT-PCR

83

miRNA

Re

lati

ve

Ch

an

ge



0

5

10

15

20

25


miRNA

Rela

tive C

han

ge

FlexmiR v2

qRT-PCR

LNP-miRNA

qPCR

84



0

5000

10000

15000

20000

25000

30000

hsa-m

iR-1

07

hsa-m

iR-1

47

hsa-m

iR-1

81a

hsa-m

iR-1

92

hsa-m

iR-1

9b

hsa-m

iR-2

20a

hsa-m

iR-2

1hsa-m

iR-2

18

hsa-le

t-7c

hsa-m

iR-1

00

hsa-m

iR-1

25b

hsa-m

iR-

hsa-m

iR-2

05

hsa-m

iR-2

21

hsa-m

iR-2

00a

hsa-m

iR-2

00c

hsa-m

iR-2

06

hsa-m

iR-1

0a

hsa-m

iR-1

6hsa-m

iR-9

hsa-m

iR-4

24

hsa-m

iR-2

0a

hsa-m

iR-2

0b

hsa-le

t-7a

hsa-m

iR-9

5hsa-m

iR-1

36

hsa-m

iR-1

95

hsa-m

iR-2

10

hsa-m

iR-2

12

hsa-m

iR-

hsa-m

iR-1

7hsa-m

iR-1

22

hsa-m

iR-1

43

hsa-m

iR-1

45

hsa-m

ir-1

06a

hsa-m

iR-

hsa-m

iR-1

0b

hsa-m

iR-1

hsa-m

iR-2

23

hsa-m

iR-1

81b

hsa-le

t-155

hsa-le

t-7g

hsa-m

iR-7

ihsa-m

iR-9

6hsa-m

iR-1

37

hsa-m

iR-1

85

hsa-m

iR-1

38

hsa-m

iR-1

33a

hsa-m

iR-1

33b

hsa-m

iR-1

5a

hsa-m

iR-3

4a

hsa-m

iR-2

9a

hsa-m

iR-2

9b

hsa-m

iR-1

03

hsa-m

iR-2

22

hsa-m

iR-1

46a

hsa-m

iR-3

9cel-m

iR-3

9cel-m

iR-2

38

Regions

Ne

t S

ign

al in

ten

sit

y (

MF

I)

50ng

100ng

250ng

500ng








bull Flexible





Proteomic Assays



Nucleic Acids








or IgM





Multiplex Detection

Detection


IgG

1 2 3 4





















Y Y

Y

IgG1 IgG2

IgG3

IgG4 IgA



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Detection



Antigen

1 2 3








Y Y Y

Y

Y

Y Y

Y Y

Y Y

Y



Y


Detection

Multiplex Detection


















compounds





compounds



SAPE SAPE














SAPE SAPE


Detection

Multiplex Detection












543-550






GTPases 827 271-282














Single Plex Assays

To Luminex

Multiplex

Assays






MagPlextrade








MicroPlextrade

Microspheres 56 um





















plate



plates




of a 96 well plate




well plates



Real Examples

Arrays

And

NGS

RT-PCR


Discovery

amp

Validation-1

An

aly

tes

S

am

ple

Samples Day

10s 100s 1000s 10000s

10

100

1000

10000 3D Throughput



6 x 384 platesday

2304 samplesday

1152000 dpday


MAGPIX

50 Plex

LX200

80-100 Plex

FLEXMAP 3D

500 Plex




concentrations

Quantify binding

events

Interrogate bead

with red laser

(635 nm)

Interrogate label

with green laser

(525 nm)

Sheath Fluid



green LED (525 nm)


LED (635 nm)


CCD imager


Capture


Genomic Assays










Detection

SAPE






HPV (high-risk

mucosal anogenital)




Quint et al 2009



HPV (high-risk

mucosal anogenital)



HPV (mucosal

anogenital)










Detection

RNA

Biotinylated cDNA








1 2





1

2

TAG-1

TAG-2



o Gene expression



o miRNA analysis


applications




ANNEAL

Ligate

PCR

TAG1

TAG2

cDNA or

gDNAPCR

HYBRIDIZE

amp

LABEL

dA

dG dT

dC -

ANNEAL

amp

Extend

TAG1

TAG2

HYBRIDIZE

amp

LABEL

gDNAPCR

SNP typing CF

CYP 450 RVP


CNV etc

RVP FAST GPP FAST

HYBRIDIZE

amp

LABEL

TAG1

TAG2 ANNEAL

amp

PCR

gDNA


30



2 hours



30 minutes



30 minutes





30 minutes







methods







TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

AAAAAAA RNA

AAAAAAA RNA

AAAAAAA RNA






TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

AAAAAAA RNA

AAAAAAA RNA

AAAAAAA RNA

cDNA

cDNA

cDNA




TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

cDNA

cDNA

cDNA




LMA Probe Design



Upstream LMA Probe

cDNA


Universal Primer 1

5rsquo 3rsquo












TAG

SAPE

Detection



lt3 Hours Setup











ANNEAL

Ligate

PCR

TAG1

TAG2

cDNA or

gDNAPCR

HYBRIDIZE

amp

LABEL





Upstream LMA Probes

T

Universal Primer 1

5rsquo

A

5rsquo

G



SNP

T



T

T


PCR Amplification

UP 1

P

No Amplification

B-UP 2



Universal Prime mix

UP 1

P

B-UP 2

UP 1

P

B-UP 2

Ligate

T T







TAG

SAPE

Detection



Bead Hyb 80 minutes







Incubation








Targeted PCR




Upstream LDR Probes

A T

G



SNP

A

A



A


Multiple

Ligation

cycles









TAG

SAPE

Detection






Forward primers

gDNA


5rsquo

3rsquo


A

T

5rsquo

3rsquo

G





Targeted PCR



A

5rsquo

3rsquo

G

5rsquo

3rsquo

T




A

5rsquo

3rsquo

G 5rsquo

3rsquo

T








xTAG


Detection

SAPE










xTAG primer

gDNA


Biotinylated primer

5rsquo 3rsquo

3rsquo

dATP dTTP dGTP dCTP

= Carbon Spacer











Detection

Sequence Detection

TAG


















Workflow

75



2 hours



30 minutes



30 minutes





30 minutes

76






0

500

1000

1500

2000

2500

3000

3500

4000


Sample

Sig

nal In

ten

sit

y (

MF

I)

let-7c

Let-7a

77


miR-107 vs miR-103

0

1000

2000

3000

4000

5000

6000


Sample

Sig

na

l In

ten

sit

y (

MF

I)

miR-103

miR-107









0

1000

2000

3000

4000

5000

6000

7000

8000

9000

10000

hsa-m

iR-1

07

hsa-m

iR-1

47

hsa-m

iR-1

81a

hsa-m

iR-1

92

hsa-m

iR-1

9b

hsa-m

iR-2

20a

hsa-m

iR-2

1hsa-m

iR-2

18

hsa-le

t-7c

hsa-m

iR-1

00

hsa-m

iR-1

25b

hsa-m

iR-

hsa-m

iR-2

05

hsa-m

iR-2

21

hsa-m

iR-2

00a

hsa-m

iR-2

00c

hsa-m

iR-2

06

hsa-m

iR-1

0a

hsa-m

iR-1

6

hsa-m

iR-9

hsa-m

iR-4

24

hsa-m

iR-2

0a

hsa-m

iR-2

0b

hsa-le

t-7a

hsa-m

iR-9

5hsa-m

iR-1

36

hsa-m

iR-1

95

hsa-m

iR-2

10

hsa-m

iR-2

12

hsa-m

iR-

hsa-m

iR-1

7

hsa-m

iR-1

22

hsa-m

iR-1

43

hsa-m

iR-1

45

hsa-m

iR-1

06a

hsa-m

iR-

hsa-m

iR-1

0b

hsa-m

iR-1

hsa-m

iR-2

23

hsa-m

iR-1

81b

hsa-m

iR-1

55

hsa-le

t-7g

hsa-le

t-7i

hsa-m

iR-9

6

hsa-m

iR-1

37

hsa-m

iR-1

85

hsa-m

iR-1

38

hsa-m

iR-1

98

hsa-m

iR-1

33a

hsa-m

iR-1

33b

hsa-m

iR-1

5a

hsa-m

iR-3

4a

hsa-m

iR-2

9a

hsa-m

iR-2

9b

hsa-m

iR-1

03

hsa-m

iR-2

22

hsa-m

iR-1

46a

cel-m

iR-3

9cel-m

iR-5

4

cel-m

iR-2

38

Regions

Ne

t S

ign

al In

ten

sit

y (

MF

I)

106a

17


Total RNA Samples

53-Plex Assay

miR-9 miR-122 miR-1

0

5000

10000

15000

20000

25000

Samples

Ne

t S

ign

al In

ten

sit

y (

MF

I)

miR-9

miR-122

miR-1






289704

12431797

4050

7044

12899

18030

24973

23884

21852

19249

11326

6573

3852

2471

0

5000

10000

15000

20000

25000

30000


Input Amount

Ne

t S

ign

al In

ten

sit

y (

MF

I)

200 amol


81


0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000



Ne

t S

ign

al in

ten

sit

y (

MF

I)

82

miR-133a

000

020

040

060

080

100

120


Total RNA Samples

Rela

tive C

han

ge t

o H

eart

FlexmiR v2

qRT-PCR

Tissue

Re

lati

ve

Ch

an

ge


LNP-miRNA

qRT-PCR

83

miRNA

Re

lati

ve

Ch

an

ge



0

5

10

15

20

25


miRNA

Rela

tive C

han

ge

FlexmiR v2

qRT-PCR

LNP-miRNA

qPCR

84



0

5000

10000

15000

20000

25000

30000

hsa-m

iR-1

07

hsa-m

iR-1

47

hsa-m

iR-1

81a

hsa-m

iR-1

92

hsa-m

iR-1

9b

hsa-m

iR-2

20a

hsa-m

iR-2

1hsa-m

iR-2

18

hsa-le

t-7c

hsa-m

iR-1

00

hsa-m

iR-1

25b

hsa-m

iR-

hsa-m

iR-2

05

hsa-m

iR-2

21

hsa-m

iR-2

00a

hsa-m

iR-2

00c

hsa-m

iR-2

06

hsa-m

iR-1

0a

hsa-m

iR-1

6hsa-m

iR-9

hsa-m

iR-4

24

hsa-m

iR-2

0a

hsa-m

iR-2

0b

hsa-le

t-7a

hsa-m

iR-9

5hsa-m

iR-1

36

hsa-m

iR-1

95

hsa-m

iR-2

10

hsa-m

iR-2

12

hsa-m

iR-

hsa-m

iR-1

7hsa-m

iR-1

22

hsa-m

iR-1

43

hsa-m

iR-1

45

hsa-m

ir-1

06a

hsa-m

iR-

hsa-m

iR-1

0b

hsa-m

iR-1

hsa-m

iR-2

23

hsa-m

iR-1

81b

hsa-le

t-155

hsa-le

t-7g

hsa-m

iR-7

ihsa-m

iR-9

6hsa-m

iR-1

37

hsa-m

iR-1

85

hsa-m

iR-1

38

hsa-m

iR-1

33a

hsa-m

iR-1

33b

hsa-m

iR-1

5a

hsa-m

iR-3

4a

hsa-m

iR-2

9a

hsa-m

iR-2

9b

hsa-m

iR-1

03

hsa-m

iR-2

22

hsa-m

iR-1

46a

hsa-m

iR-3

9cel-m

iR-3

9cel-m

iR-2

38

Regions

Ne

t S

ign

al in

ten

sit

y (

MF

I)

50ng

100ng

250ng

500ng








bull Flexible





Proteomic Assays



Nucleic Acids








or IgM





Multiplex Detection

Detection


IgG

1 2 3 4





















Y Y

Y

IgG1 IgG2

IgG3

IgG4 IgA



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Detection



Antigen

1 2 3








Y Y Y

Y

Y

Y Y

Y Y

Y Y

Y



Y


Detection

Multiplex Detection


















compounds





compounds



SAPE SAPE














SAPE SAPE


Detection

Multiplex Detection












543-550






GTPases 827 271-282














Single Plex Assays

To Luminex

Multiplex

Assays





MagPlextrade








MicroPlextrade

Microspheres 56 um





















plate



plates




of a 96 well plate




well plates



Real Examples

Arrays

And

NGS

RT-PCR


Discovery

amp

Validation-1

An

aly

tes

S

am

ple

Samples Day

10s 100s 1000s 10000s

10

100

1000

10000 3D Throughput



6 x 384 platesday

2304 samplesday

1152000 dpday


MAGPIX

50 Plex

LX200

80-100 Plex

FLEXMAP 3D

500 Plex




concentrations

Quantify binding

events

Interrogate bead

with red laser

(635 nm)

Interrogate label

with green laser

(525 nm)

Sheath Fluid



green LED (525 nm)


LED (635 nm)


CCD imager


Capture


Genomic Assays










Detection

SAPE






HPV (high-risk

mucosal anogenital)




Quint et al 2009



HPV (high-risk

mucosal anogenital)



HPV (mucosal

anogenital)










Detection

RNA

Biotinylated cDNA








1 2





1

2

TAG-1

TAG-2



o Gene expression



o miRNA analysis


applications




ANNEAL

Ligate

PCR

TAG1

TAG2

cDNA or

gDNAPCR

HYBRIDIZE

amp

LABEL

dA

dG dT

dC -

ANNEAL

amp

Extend

TAG1

TAG2

HYBRIDIZE

amp

LABEL

gDNAPCR

SNP typing CF

CYP 450 RVP


CNV etc

RVP FAST GPP FAST

HYBRIDIZE

amp

LABEL

TAG1

TAG2 ANNEAL

amp

PCR

gDNA


30



2 hours



30 minutes



30 minutes





30 minutes







methods







TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

AAAAAAA RNA

AAAAAAA RNA

AAAAAAA RNA






TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

AAAAAAA RNA

AAAAAAA RNA

AAAAAAA RNA

cDNA

cDNA

cDNA




TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

cDNA

cDNA

cDNA




LMA Probe Design



Upstream LMA Probe

cDNA


Universal Primer 1

5rsquo 3rsquo












TAG

SAPE

Detection



lt3 Hours Setup











ANNEAL

Ligate

PCR

TAG1

TAG2

cDNA or

gDNAPCR

HYBRIDIZE

amp

LABEL





Upstream LMA Probes

T

Universal Primer 1

5rsquo

A

5rsquo

G



SNP

T



T

T


PCR Amplification

UP 1

P

No Amplification

B-UP 2



Universal Prime mix

UP 1

P

B-UP 2

UP 1

P

B-UP 2

Ligate

T T







TAG

SAPE

Detection



Bead Hyb 80 minutes







Incubation








Targeted PCR




Upstream LDR Probes

A T

G



SNP

A

A



A


Multiple

Ligation

cycles









TAG

SAPE

Detection






Forward primers

gDNA


5rsquo

3rsquo


A

T

5rsquo

3rsquo

G





Targeted PCR



A

5rsquo

3rsquo

G

5rsquo

3rsquo

T




A

5rsquo

3rsquo

G 5rsquo

3rsquo

T








xTAG


Detection

SAPE










xTAG primer

gDNA


Biotinylated primer

5rsquo 3rsquo

3rsquo

dATP dTTP dGTP dCTP

= Carbon Spacer











Detection

Sequence Detection

TAG


















Workflow

75



2 hours



30 minutes



30 minutes





30 minutes

76






0

500

1000

1500

2000

2500

3000

3500

4000


Sample

Sig

nal In

ten

sit

y (

MF

I)

let-7c

Let-7a

77


miR-107 vs miR-103

0

1000

2000

3000

4000

5000

6000


Sample

Sig

na

l In

ten

sit

y (

MF

I)

miR-103

miR-107









0

1000

2000

3000

4000

5000

6000

7000

8000

9000

10000

hsa-m

iR-1

07

hsa-m

iR-1

47

hsa-m

iR-1

81a

hsa-m

iR-1

92

hsa-m

iR-1

9b

hsa-m

iR-2

20a

hsa-m

iR-2

1hsa-m

iR-2

18

hsa-le

t-7c

hsa-m

iR-1

00

hsa-m

iR-1

25b

hsa-m

iR-

hsa-m

iR-2

05

hsa-m

iR-2

21

hsa-m

iR-2

00a

hsa-m

iR-2

00c

hsa-m

iR-2

06

hsa-m

iR-1

0a

hsa-m

iR-1

6

hsa-m

iR-9

hsa-m

iR-4

24

hsa-m

iR-2

0a

hsa-m

iR-2

0b

hsa-le

t-7a

hsa-m

iR-9

5hsa-m

iR-1

36

hsa-m

iR-1

95

hsa-m

iR-2

10

hsa-m

iR-2

12

hsa-m

iR-

hsa-m

iR-1

7

hsa-m

iR-1

22

hsa-m

iR-1

43

hsa-m

iR-1

45

hsa-m

iR-1

06a

hsa-m

iR-

hsa-m

iR-1

0b

hsa-m

iR-1

hsa-m

iR-2

23

hsa-m

iR-1

81b

hsa-m

iR-1

55

hsa-le

t-7g

hsa-le

t-7i

hsa-m

iR-9

6

hsa-m

iR-1

37

hsa-m

iR-1

85

hsa-m

iR-1

38

hsa-m

iR-1

98

hsa-m

iR-1

33a

hsa-m

iR-1

33b

hsa-m

iR-1

5a

hsa-m

iR-3

4a

hsa-m

iR-2

9a

hsa-m

iR-2

9b

hsa-m

iR-1

03

hsa-m

iR-2

22

hsa-m

iR-1

46a

cel-m

iR-3

9cel-m

iR-5

4

cel-m

iR-2

38

Regions

Ne

t S

ign

al In

ten

sit

y (

MF

I)

106a

17


Total RNA Samples

53-Plex Assay

miR-9 miR-122 miR-1

0

5000

10000

15000

20000

25000

Samples

Ne

t S

ign

al In

ten

sit

y (

MF

I)

miR-9

miR-122

miR-1






289704

12431797

4050

7044

12899

18030

24973

23884

21852

19249

11326

6573

3852

2471

0

5000

10000

15000

20000

25000

30000


Input Amount

Ne

t S

ign

al In

ten

sit

y (

MF

I)

200 amol


81


0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000



Ne

t S

ign

al in

ten

sit

y (

MF

I)

82

miR-133a

000

020

040

060

080

100

120


Total RNA Samples

Rela

tive C

han

ge t

o H

eart

FlexmiR v2

qRT-PCR

Tissue

Re

lati

ve

Ch

an

ge


LNP-miRNA

qRT-PCR

83

miRNA

Re

lati

ve

Ch

an

ge



0

5

10

15

20

25


miRNA

Rela

tive C

han

ge

FlexmiR v2

qRT-PCR

LNP-miRNA

qPCR

84



0

5000

10000

15000

20000

25000

30000

hsa-m

iR-1

07

hsa-m

iR-1

47

hsa-m

iR-1

81a

hsa-m

iR-1

92

hsa-m

iR-1

9b

hsa-m

iR-2

20a

hsa-m

iR-2

1hsa-m

iR-2

18

hsa-le

t-7c

hsa-m

iR-1

00

hsa-m

iR-1

25b

hsa-m

iR-

hsa-m

iR-2

05

hsa-m

iR-2

21

hsa-m

iR-2

00a

hsa-m

iR-2

00c

hsa-m

iR-2

06

hsa-m

iR-1

0a

hsa-m

iR-1

6hsa-m

iR-9

hsa-m

iR-4

24

hsa-m

iR-2

0a

hsa-m

iR-2

0b

hsa-le

t-7a

hsa-m

iR-9

5hsa-m

iR-1

36

hsa-m

iR-1

95

hsa-m

iR-2

10

hsa-m

iR-2

12

hsa-m

iR-

hsa-m

iR-1

7hsa-m

iR-1

22

hsa-m

iR-1

43

hsa-m

iR-1

45

hsa-m

ir-1

06a

hsa-m

iR-

hsa-m

iR-1

0b

hsa-m

iR-1

hsa-m

iR-2

23

hsa-m

iR-1

81b

hsa-le

t-155

hsa-le

t-7g

hsa-m

iR-7

ihsa-m

iR-9

6hsa-m

iR-1

37

hsa-m

iR-1

85

hsa-m

iR-1

38

hsa-m

iR-1

33a

hsa-m

iR-1

33b

hsa-m

iR-1

5a

hsa-m

iR-3

4a

hsa-m

iR-2

9a

hsa-m

iR-2

9b

hsa-m

iR-1

03

hsa-m

iR-2

22

hsa-m

iR-1

46a

hsa-m

iR-3

9cel-m

iR-3

9cel-m

iR-2

38

Regions

Ne

t S

ign

al in

ten

sit

y (

MF

I)

50ng

100ng

250ng

500ng








bull Flexible





Proteomic Assays



Nucleic Acids








or IgM





Multiplex Detection

Detection


IgG

1 2 3 4





















Y Y

Y

IgG1 IgG2

IgG3

IgG4 IgA



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Detection



Antigen

1 2 3








Y Y Y

Y

Y

Y Y

Y Y

Y Y

Y



Y


Detection

Multiplex Detection


















compounds





compounds



SAPE SAPE














SAPE SAPE


Detection

Multiplex Detection












543-550






GTPases 827 271-282














Single Plex Assays

To Luminex

Multiplex

Assays























plate



plates




of a 96 well plate




well plates



Real Examples

Arrays

And

NGS

RT-PCR


Discovery

amp

Validation-1

An

aly

tes

S

am

ple

Samples Day

10s 100s 1000s 10000s

10

100

1000

10000 3D Throughput



6 x 384 platesday

2304 samplesday

1152000 dpday


MAGPIX

50 Plex

LX200

80-100 Plex

FLEXMAP 3D

500 Plex




concentrations

Quantify binding

events

Interrogate bead

with red laser

(635 nm)

Interrogate label

with green laser

(525 nm)

Sheath Fluid



green LED (525 nm)


LED (635 nm)


CCD imager


Capture


Genomic Assays










Detection

SAPE






HPV (high-risk

mucosal anogenital)




Quint et al 2009



HPV (high-risk

mucosal anogenital)



HPV (mucosal

anogenital)










Detection

RNA

Biotinylated cDNA








1 2





1

2

TAG-1

TAG-2



o Gene expression



o miRNA analysis


applications




ANNEAL

Ligate

PCR

TAG1

TAG2

cDNA or

gDNAPCR

HYBRIDIZE

amp

LABEL

dA

dG dT

dC -

ANNEAL

amp

Extend

TAG1

TAG2

HYBRIDIZE

amp

LABEL

gDNAPCR

SNP typing CF

CYP 450 RVP


CNV etc

RVP FAST GPP FAST

HYBRIDIZE

amp

LABEL

TAG1

TAG2 ANNEAL

amp

PCR

gDNA


30



2 hours



30 minutes



30 minutes





30 minutes







methods







TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

AAAAAAA RNA

AAAAAAA RNA

AAAAAAA RNA






TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

AAAAAAA RNA

AAAAAAA RNA

AAAAAAA RNA

cDNA

cDNA

cDNA




TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

cDNA

cDNA

cDNA




LMA Probe Design



Upstream LMA Probe

cDNA


Universal Primer 1

5rsquo 3rsquo












TAG

SAPE

Detection



lt3 Hours Setup











ANNEAL

Ligate

PCR

TAG1

TAG2

cDNA or

gDNAPCR

HYBRIDIZE

amp

LABEL





Upstream LMA Probes

T

Universal Primer 1

5rsquo

A

5rsquo

G



SNP

T



T

T


PCR Amplification

UP 1

P

No Amplification

B-UP 2



Universal Prime mix

UP 1

P

B-UP 2

UP 1

P

B-UP 2

Ligate

T T







TAG

SAPE

Detection



Bead Hyb 80 minutes







Incubation








Targeted PCR




Upstream LDR Probes

A T

G



SNP

A

A



A


Multiple

Ligation

cycles









TAG

SAPE

Detection






Forward primers

gDNA


5rsquo

3rsquo


A

T

5rsquo

3rsquo

G





Targeted PCR



A

5rsquo

3rsquo

G

5rsquo

3rsquo

T




A

5rsquo

3rsquo

G 5rsquo

3rsquo

T








xTAG


Detection

SAPE










xTAG primer

gDNA


Biotinylated primer

5rsquo 3rsquo

3rsquo

dATP dTTP dGTP dCTP

= Carbon Spacer











Detection

Sequence Detection

TAG


















Workflow

75



2 hours



30 minutes



30 minutes





30 minutes

76






0

500

1000

1500

2000

2500

3000

3500

4000


Sample

Sig

nal In

ten

sit

y (

MF

I)

let-7c

Let-7a

77


miR-107 vs miR-103

0

1000

2000

3000

4000

5000

6000


Sample

Sig

na

l In

ten

sit

y (

MF

I)

miR-103

miR-107









0

1000

2000

3000

4000

5000

6000

7000

8000

9000

10000

hsa-m

iR-1

07

hsa-m

iR-1

47

hsa-m

iR-1

81a

hsa-m

iR-1

92

hsa-m

iR-1

9b

hsa-m

iR-2

20a

hsa-m

iR-2

1hsa-m

iR-2

18

hsa-le

t-7c

hsa-m

iR-1

00

hsa-m

iR-1

25b

hsa-m

iR-

hsa-m

iR-2

05

hsa-m

iR-2

21

hsa-m

iR-2

00a

hsa-m

iR-2

00c

hsa-m

iR-2

06

hsa-m

iR-1

0a

hsa-m

iR-1

6

hsa-m

iR-9

hsa-m

iR-4

24

hsa-m

iR-2

0a

hsa-m

iR-2

0b

hsa-le

t-7a

hsa-m

iR-9

5hsa-m

iR-1

36

hsa-m

iR-1

95

hsa-m

iR-2

10

hsa-m

iR-2

12

hsa-m

iR-

hsa-m

iR-1

7

hsa-m

iR-1

22

hsa-m

iR-1

43

hsa-m

iR-1

45

hsa-m

iR-1

06a

hsa-m

iR-

hsa-m

iR-1

0b

hsa-m

iR-1

hsa-m

iR-2

23

hsa-m

iR-1

81b

hsa-m

iR-1

55

hsa-le

t-7g

hsa-le

t-7i

hsa-m

iR-9

6

hsa-m

iR-1

37

hsa-m

iR-1

85

hsa-m

iR-1

38

hsa-m

iR-1

98

hsa-m

iR-1

33a

hsa-m

iR-1

33b

hsa-m

iR-1

5a

hsa-m

iR-3

4a

hsa-m

iR-2

9a

hsa-m

iR-2

9b

hsa-m

iR-1

03

hsa-m

iR-2

22

hsa-m

iR-1

46a

cel-m

iR-3

9cel-m

iR-5

4

cel-m

iR-2

38

Regions

Ne

t S

ign

al In

ten

sit

y (

MF

I)

106a

17


Total RNA Samples

53-Plex Assay

miR-9 miR-122 miR-1

0

5000

10000

15000

20000

25000

Samples

Ne

t S

ign

al In

ten

sit

y (

MF

I)

miR-9

miR-122

miR-1






289704

12431797

4050

7044

12899

18030

24973

23884

21852

19249

11326

6573

3852

2471

0

5000

10000

15000

20000

25000

30000


Input Amount

Ne

t S

ign

al In

ten

sit

y (

MF

I)

200 amol


81


0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000



Ne

t S

ign

al in

ten

sit

y (

MF

I)

82

miR-133a

000

020

040

060

080

100

120


Total RNA Samples

Rela

tive C

han

ge t

o H

eart

FlexmiR v2

qRT-PCR

Tissue

Re

lati

ve

Ch

an

ge


LNP-miRNA

qRT-PCR

83

miRNA

Re

lati

ve

Ch

an

ge



0

5

10

15

20

25


miRNA

Rela

tive C

han

ge

FlexmiR v2

qRT-PCR

LNP-miRNA

qPCR

84



0

5000

10000

15000

20000

25000

30000

hsa-m

iR-1

07

hsa-m

iR-1

47

hsa-m

iR-1

81a

hsa-m

iR-1

92

hsa-m

iR-1

9b

hsa-m

iR-2

20a

hsa-m

iR-2

1hsa-m

iR-2

18

hsa-le

t-7c

hsa-m

iR-1

00

hsa-m

iR-1

25b

hsa-m

iR-

hsa-m

iR-2

05

hsa-m

iR-2

21

hsa-m

iR-2

00a

hsa-m

iR-2

00c

hsa-m

iR-2

06

hsa-m

iR-1

0a

hsa-m

iR-1

6hsa-m

iR-9

hsa-m

iR-4

24

hsa-m

iR-2

0a

hsa-m

iR-2

0b

hsa-le

t-7a

hsa-m

iR-9

5hsa-m

iR-1

36

hsa-m

iR-1

95

hsa-m

iR-2

10

hsa-m

iR-2

12

hsa-m

iR-

hsa-m

iR-1

7hsa-m

iR-1

22

hsa-m

iR-1

43

hsa-m

iR-1

45

hsa-m

ir-1

06a

hsa-m

iR-

hsa-m

iR-1

0b

hsa-m

iR-1

hsa-m

iR-2

23

hsa-m

iR-1

81b

hsa-le

t-155

hsa-le

t-7g

hsa-m

iR-7

ihsa-m

iR-9

6hsa-m

iR-1

37

hsa-m

iR-1

85

hsa-m

iR-1

38

hsa-m

iR-1

33a

hsa-m

iR-1

33b

hsa-m

iR-1

5a

hsa-m

iR-3

4a

hsa-m

iR-2

9a

hsa-m

iR-2

9b

hsa-m

iR-1

03

hsa-m

iR-2

22

hsa-m

iR-1

46a

hsa-m

iR-3

9cel-m

iR-3

9cel-m

iR-2

38

Regions

Ne

t S

ign

al in

ten

sit

y (

MF

I)

50ng

100ng

250ng

500ng








bull Flexible





Proteomic Assays



Nucleic Acids








or IgM





Multiplex Detection

Detection


IgG

1 2 3 4





















Y Y

Y

IgG1 IgG2

IgG3

IgG4 IgA



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Detection



Antigen

1 2 3








Y Y Y

Y

Y

Y Y

Y Y

Y Y

Y



Y


Detection

Multiplex Detection


















compounds





compounds



SAPE SAPE














SAPE SAPE


Detection

Multiplex Detection












543-550






GTPases 827 271-282














Single Plex Assays

To Luminex

Multiplex

Assays














plate



plates




of a 96 well plate




well plates



Real Examples

Arrays

And

NGS

RT-PCR


Discovery

amp

Validation-1

An

aly

tes

S

am

ple

Samples Day

10s 100s 1000s 10000s

10

100

1000

10000 3D Throughput



6 x 384 platesday

2304 samplesday

1152000 dpday


MAGPIX

50 Plex

LX200

80-100 Plex

FLEXMAP 3D

500 Plex




concentrations

Quantify binding

events

Interrogate bead

with red laser

(635 nm)

Interrogate label

with green laser

(525 nm)

Sheath Fluid



green LED (525 nm)


LED (635 nm)


CCD imager


Capture


Genomic Assays










Detection

SAPE






HPV (high-risk

mucosal anogenital)




Quint et al 2009



HPV (high-risk

mucosal anogenital)



HPV (mucosal

anogenital)










Detection

RNA

Biotinylated cDNA








1 2





1

2

TAG-1

TAG-2



o Gene expression



o miRNA analysis


applications




ANNEAL

Ligate

PCR

TAG1

TAG2

cDNA or

gDNAPCR

HYBRIDIZE

amp

LABEL

dA

dG dT

dC -

ANNEAL

amp

Extend

TAG1

TAG2

HYBRIDIZE

amp

LABEL

gDNAPCR

SNP typing CF

CYP 450 RVP


CNV etc

RVP FAST GPP FAST

HYBRIDIZE

amp

LABEL

TAG1

TAG2 ANNEAL

amp

PCR

gDNA


30



2 hours



30 minutes



30 minutes





30 minutes







methods







TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

AAAAAAA RNA

AAAAAAA RNA

AAAAAAA RNA






TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

AAAAAAA RNA

AAAAAAA RNA

AAAAAAA RNA

cDNA

cDNA

cDNA




TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

cDNA

cDNA

cDNA




LMA Probe Design



Upstream LMA Probe

cDNA


Universal Primer 1

5rsquo 3rsquo












TAG

SAPE

Detection



lt3 Hours Setup











ANNEAL

Ligate

PCR

TAG1

TAG2

cDNA or

gDNAPCR

HYBRIDIZE

amp

LABEL





Upstream LMA Probes

T

Universal Primer 1

5rsquo

A

5rsquo

G



SNP

T



T

T


PCR Amplification

UP 1

P

No Amplification

B-UP 2



Universal Prime mix

UP 1

P

B-UP 2

UP 1

P

B-UP 2

Ligate

T T







TAG

SAPE

Detection



Bead Hyb 80 minutes







Incubation








Targeted PCR




Upstream LDR Probes

A T

G



SNP

A

A



A


Multiple

Ligation

cycles









TAG

SAPE

Detection






Forward primers

gDNA


5rsquo

3rsquo


A

T

5rsquo

3rsquo

G





Targeted PCR



A

5rsquo

3rsquo

G

5rsquo

3rsquo

T




A

5rsquo

3rsquo

G 5rsquo

3rsquo

T








xTAG


Detection

SAPE










xTAG primer

gDNA


Biotinylated primer

5rsquo 3rsquo

3rsquo

dATP dTTP dGTP dCTP

= Carbon Spacer











Detection

Sequence Detection

TAG


















Workflow

75



2 hours



30 minutes



30 minutes





30 minutes

76






0

500

1000

1500

2000

2500

3000

3500

4000


Sample

Sig

nal In

ten

sit

y (

MF

I)

let-7c

Let-7a

77


miR-107 vs miR-103

0

1000

2000

3000

4000

5000

6000


Sample

Sig

na

l In

ten

sit

y (

MF

I)

miR-103

miR-107









0

1000

2000

3000

4000

5000

6000

7000

8000

9000

10000

hsa-m

iR-1

07

hsa-m

iR-1

47

hsa-m

iR-1

81a

hsa-m

iR-1

92

hsa-m

iR-1

9b

hsa-m

iR-2

20a

hsa-m

iR-2

1hsa-m

iR-2

18

hsa-le

t-7c

hsa-m

iR-1

00

hsa-m

iR-1

25b

hsa-m

iR-

hsa-m

iR-2

05

hsa-m

iR-2

21

hsa-m

iR-2

00a

hsa-m

iR-2

00c

hsa-m

iR-2

06

hsa-m

iR-1

0a

hsa-m

iR-1

6

hsa-m

iR-9

hsa-m

iR-4

24

hsa-m

iR-2

0a

hsa-m

iR-2

0b

hsa-le

t-7a

hsa-m

iR-9

5hsa-m

iR-1

36

hsa-m

iR-1

95

hsa-m

iR-2

10

hsa-m

iR-2

12

hsa-m

iR-

hsa-m

iR-1

7

hsa-m

iR-1

22

hsa-m

iR-1

43

hsa-m

iR-1

45

hsa-m

iR-1

06a

hsa-m

iR-

hsa-m

iR-1

0b

hsa-m

iR-1

hsa-m

iR-2

23

hsa-m

iR-1

81b

hsa-m

iR-1

55

hsa-le

t-7g

hsa-le

t-7i

hsa-m

iR-9

6

hsa-m

iR-1

37

hsa-m

iR-1

85

hsa-m

iR-1

38

hsa-m

iR-1

98

hsa-m

iR-1

33a

hsa-m

iR-1

33b

hsa-m

iR-1

5a

hsa-m

iR-3

4a

hsa-m

iR-2

9a

hsa-m

iR-2

9b

hsa-m

iR-1

03

hsa-m

iR-2

22

hsa-m

iR-1

46a

cel-m

iR-3

9cel-m

iR-5

4

cel-m

iR-2

38

Regions

Ne

t S

ign

al In

ten

sit

y (

MF

I)

106a

17


Total RNA Samples

53-Plex Assay

miR-9 miR-122 miR-1

0

5000

10000

15000

20000

25000

Samples

Ne

t S

ign

al In

ten

sit

y (

MF

I)

miR-9

miR-122

miR-1






289704

12431797

4050

7044

12899

18030

24973

23884

21852

19249

11326

6573

3852

2471

0

5000

10000

15000

20000

25000

30000


Input Amount

Ne

t S

ign

al In

ten

sit

y (

MF

I)

200 amol


81


0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000



Ne

t S

ign

al in

ten

sit

y (

MF

I)

82

miR-133a

000

020

040

060

080

100

120


Total RNA Samples

Rela

tive C

han

ge t

o H

eart

FlexmiR v2

qRT-PCR

Tissue

Re

lati

ve

Ch

an

ge


LNP-miRNA

qRT-PCR

83

miRNA

Re

lati

ve

Ch

an

ge



0

5

10

15

20

25


miRNA

Rela

tive C

han

ge

FlexmiR v2

qRT-PCR

LNP-miRNA

qPCR

84



0

5000

10000

15000

20000

25000

30000

hsa-m

iR-1

07

hsa-m

iR-1

47

hsa-m

iR-1

81a

hsa-m

iR-1

92

hsa-m

iR-1

9b

hsa-m

iR-2

20a

hsa-m

iR-2

1hsa-m

iR-2

18

hsa-le

t-7c

hsa-m

iR-1

00

hsa-m

iR-1

25b

hsa-m

iR-

hsa-m

iR-2

05

hsa-m

iR-2

21

hsa-m

iR-2

00a

hsa-m

iR-2

00c

hsa-m

iR-2

06

hsa-m

iR-1

0a

hsa-m

iR-1

6hsa-m

iR-9

hsa-m

iR-4

24

hsa-m

iR-2

0a

hsa-m

iR-2

0b

hsa-le

t-7a

hsa-m

iR-9

5hsa-m

iR-1

36

hsa-m

iR-1

95

hsa-m

iR-2

10

hsa-m

iR-2

12

hsa-m

iR-

hsa-m

iR-1

7hsa-m

iR-1

22

hsa-m

iR-1

43

hsa-m

iR-1

45

hsa-m

ir-1

06a

hsa-m

iR-

hsa-m

iR-1

0b

hsa-m

iR-1

hsa-m

iR-2

23

hsa-m

iR-1

81b

hsa-le

t-155

hsa-le

t-7g

hsa-m

iR-7

ihsa-m

iR-9

6hsa-m

iR-1

37

hsa-m

iR-1

85

hsa-m

iR-1

38

hsa-m

iR-1

33a

hsa-m

iR-1

33b

hsa-m

iR-1

5a

hsa-m

iR-3

4a

hsa-m

iR-2

9a

hsa-m

iR-2

9b

hsa-m

iR-1

03

hsa-m

iR-2

22

hsa-m

iR-1

46a

hsa-m

iR-3

9cel-m

iR-3

9cel-m

iR-2

38

Regions

Ne

t S

ign

al in

ten

sit

y (

MF

I)

50ng

100ng

250ng

500ng








bull Flexible





Proteomic Assays



Nucleic Acids








or IgM





Multiplex Detection

Detection


IgG

1 2 3 4





















Y Y

Y

IgG1 IgG2

IgG3

IgG4 IgA



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Detection



Antigen

1 2 3








Y Y Y

Y

Y

Y Y

Y Y

Y Y

Y



Y


Detection

Multiplex Detection


















compounds





compounds



SAPE SAPE














SAPE SAPE


Detection

Multiplex Detection












543-550






GTPases 827 271-282














Single Plex Assays

To Luminex

Multiplex

Assays










plate



plates




of a 96 well plate




well plates



Real Examples

Arrays

And

NGS

RT-PCR


Discovery

amp

Validation-1

An

aly

tes

S

am

ple

Samples Day

10s 100s 1000s 10000s

10

100

1000

10000 3D Throughput



6 x 384 platesday

2304 samplesday

1152000 dpday


MAGPIX

50 Plex

LX200

80-100 Plex

FLEXMAP 3D

500 Plex




concentrations

Quantify binding

events

Interrogate bead

with red laser

(635 nm)

Interrogate label

with green laser

(525 nm)

Sheath Fluid



green LED (525 nm)


LED (635 nm)


CCD imager


Capture


Genomic Assays










Detection

SAPE






HPV (high-risk

mucosal anogenital)




Quint et al 2009



HPV (high-risk

mucosal anogenital)



HPV (mucosal

anogenital)










Detection

RNA

Biotinylated cDNA








1 2





1

2

TAG-1

TAG-2



o Gene expression



o miRNA analysis


applications




ANNEAL

Ligate

PCR

TAG1

TAG2

cDNA or

gDNAPCR

HYBRIDIZE

amp

LABEL

dA

dG dT

dC -

ANNEAL

amp

Extend

TAG1

TAG2

HYBRIDIZE

amp

LABEL

gDNAPCR

SNP typing CF

CYP 450 RVP


CNV etc

RVP FAST GPP FAST

HYBRIDIZE

amp

LABEL

TAG1

TAG2 ANNEAL

amp

PCR

gDNA


30



2 hours



30 minutes



30 minutes





30 minutes







methods







TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

AAAAAAA RNA

AAAAAAA RNA

AAAAAAA RNA






TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

AAAAAAA RNA

AAAAAAA RNA

AAAAAAA RNA

cDNA

cDNA

cDNA




TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

cDNA

cDNA

cDNA




LMA Probe Design



Upstream LMA Probe

cDNA


Universal Primer 1

5rsquo 3rsquo












TAG

SAPE

Detection



lt3 Hours Setup











ANNEAL

Ligate

PCR

TAG1

TAG2

cDNA or

gDNAPCR

HYBRIDIZE

amp

LABEL





Upstream LMA Probes

T

Universal Primer 1

5rsquo

A

5rsquo

G



SNP

T



T

T


PCR Amplification

UP 1

P

No Amplification

B-UP 2



Universal Prime mix

UP 1

P

B-UP 2

UP 1

P

B-UP 2

Ligate

T T







TAG

SAPE

Detection



Bead Hyb 80 minutes







Incubation








Targeted PCR




Upstream LDR Probes

A T

G



SNP

A

A



A


Multiple

Ligation

cycles









TAG

SAPE

Detection






Forward primers

gDNA


5rsquo

3rsquo


A

T

5rsquo

3rsquo

G





Targeted PCR



A

5rsquo

3rsquo

G

5rsquo

3rsquo

T




A

5rsquo

3rsquo

G 5rsquo

3rsquo

T








xTAG


Detection

SAPE










xTAG primer

gDNA


Biotinylated primer

5rsquo 3rsquo

3rsquo

dATP dTTP dGTP dCTP

= Carbon Spacer











Detection

Sequence Detection

TAG


















Workflow

75



2 hours



30 minutes



30 minutes





30 minutes

76






0

500

1000

1500

2000

2500

3000

3500

4000


Sample

Sig

nal In

ten

sit

y (

MF

I)

let-7c

Let-7a

77


miR-107 vs miR-103

0

1000

2000

3000

4000

5000

6000


Sample

Sig

na

l In

ten

sit

y (

MF

I)

miR-103

miR-107









0

1000

2000

3000

4000

5000

6000

7000

8000

9000

10000

hsa-m

iR-1

07

hsa-m

iR-1

47

hsa-m

iR-1

81a

hsa-m

iR-1

92

hsa-m

iR-1

9b

hsa-m

iR-2

20a

hsa-m

iR-2

1hsa-m

iR-2

18

hsa-le

t-7c

hsa-m

iR-1

00

hsa-m

iR-1

25b

hsa-m

iR-

hsa-m

iR-2

05

hsa-m

iR-2

21

hsa-m

iR-2

00a

hsa-m

iR-2

00c

hsa-m

iR-2

06

hsa-m

iR-1

0a

hsa-m

iR-1

6

hsa-m

iR-9

hsa-m

iR-4

24

hsa-m

iR-2

0a

hsa-m

iR-2

0b

hsa-le

t-7a

hsa-m

iR-9

5hsa-m

iR-1

36

hsa-m

iR-1

95

hsa-m

iR-2

10

hsa-m

iR-2

12

hsa-m

iR-

hsa-m

iR-1

7

hsa-m

iR-1

22

hsa-m

iR-1

43

hsa-m

iR-1

45

hsa-m

iR-1

06a

hsa-m

iR-

hsa-m

iR-1

0b

hsa-m

iR-1

hsa-m

iR-2

23

hsa-m

iR-1

81b

hsa-m

iR-1

55

hsa-le

t-7g

hsa-le

t-7i

hsa-m

iR-9

6

hsa-m

iR-1

37

hsa-m

iR-1

85

hsa-m

iR-1

38

hsa-m

iR-1

98

hsa-m

iR-1

33a

hsa-m

iR-1

33b

hsa-m

iR-1

5a

hsa-m

iR-3

4a

hsa-m

iR-2

9a

hsa-m

iR-2

9b

hsa-m

iR-1

03

hsa-m

iR-2

22

hsa-m

iR-1

46a

cel-m

iR-3

9cel-m

iR-5

4

cel-m

iR-2

38

Regions

Ne

t S

ign

al In

ten

sit

y (

MF

I)

106a

17


Total RNA Samples

53-Plex Assay

miR-9 miR-122 miR-1

0

5000

10000

15000

20000

25000

Samples

Ne

t S

ign

al In

ten

sit

y (

MF

I)

miR-9

miR-122

miR-1






289704

12431797

4050

7044

12899

18030

24973

23884

21852

19249

11326

6573

3852

2471

0

5000

10000

15000

20000

25000

30000


Input Amount

Ne

t S

ign

al In

ten

sit

y (

MF

I)

200 amol


81


0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000



Ne

t S

ign

al in

ten

sit

y (

MF

I)

82

miR-133a

000

020

040

060

080

100

120


Total RNA Samples

Rela

tive C

han

ge t

o H

eart

FlexmiR v2

qRT-PCR

Tissue

Re

lati

ve

Ch

an

ge


LNP-miRNA

qRT-PCR

83

miRNA

Re

lati

ve

Ch

an

ge



0

5

10

15

20

25


miRNA

Rela

tive C

han

ge

FlexmiR v2

qRT-PCR

LNP-miRNA

qPCR

84



0

5000

10000

15000

20000

25000

30000

hsa-m

iR-1

07

hsa-m

iR-1

47

hsa-m

iR-1

81a

hsa-m

iR-1

92

hsa-m

iR-1

9b

hsa-m

iR-2

20a

hsa-m

iR-2

1hsa-m

iR-2

18

hsa-le

t-7c

hsa-m

iR-1

00

hsa-m

iR-1

25b

hsa-m

iR-

hsa-m

iR-2

05

hsa-m

iR-2

21

hsa-m

iR-2

00a

hsa-m

iR-2

00c

hsa-m

iR-2

06

hsa-m

iR-1

0a

hsa-m

iR-1

6hsa-m

iR-9

hsa-m

iR-4

24

hsa-m

iR-2

0a

hsa-m

iR-2

0b

hsa-le

t-7a

hsa-m

iR-9

5hsa-m

iR-1

36

hsa-m

iR-1

95

hsa-m

iR-2

10

hsa-m

iR-2

12

hsa-m

iR-

hsa-m

iR-1

7hsa-m

iR-1

22

hsa-m

iR-1

43

hsa-m

iR-1

45

hsa-m

ir-1

06a

hsa-m

iR-

hsa-m

iR-1

0b

hsa-m

iR-1

hsa-m

iR-2

23

hsa-m

iR-1

81b

hsa-le

t-155

hsa-le

t-7g

hsa-m

iR-7

ihsa-m

iR-9

6hsa-m

iR-1

37

hsa-m

iR-1

85

hsa-m

iR-1

38

hsa-m

iR-1

33a

hsa-m

iR-1

33b

hsa-m

iR-1

5a

hsa-m

iR-3

4a

hsa-m

iR-2

9a

hsa-m

iR-2

9b

hsa-m

iR-1

03

hsa-m

iR-2

22

hsa-m

iR-1

46a

hsa-m

iR-3

9cel-m

iR-3

9cel-m

iR-2

38

Regions

Ne

t S

ign

al in

ten

sit

y (

MF

I)

50ng

100ng

250ng

500ng








bull Flexible





Proteomic Assays



Nucleic Acids








or IgM





Multiplex Detection

Detection


IgG

1 2 3 4





















Y Y

Y

IgG1 IgG2

IgG3

IgG4 IgA



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Detection



Antigen

1 2 3








Y Y Y

Y

Y

Y Y

Y Y

Y Y

Y



Y


Detection

Multiplex Detection


















compounds





compounds



SAPE SAPE














SAPE SAPE


Detection

Multiplex Detection












543-550






GTPases 827 271-282














Single Plex Assays

To Luminex

Multiplex

Assays







plate



plates




of a 96 well plate




well plates



Real Examples

Arrays

And

NGS

RT-PCR


Discovery

amp

Validation-1

An

aly

tes

S

am

ple

Samples Day

10s 100s 1000s 10000s

10

100

1000

10000 3D Throughput



6 x 384 platesday

2304 samplesday

1152000 dpday


MAGPIX

50 Plex

LX200

80-100 Plex

FLEXMAP 3D

500 Plex




concentrations

Quantify binding

events

Interrogate bead

with red laser

(635 nm)

Interrogate label

with green laser

(525 nm)

Sheath Fluid



green LED (525 nm)


LED (635 nm)


CCD imager


Capture


Genomic Assays










Detection

SAPE






HPV (high-risk

mucosal anogenital)




Quint et al 2009



HPV (high-risk

mucosal anogenital)



HPV (mucosal

anogenital)










Detection

RNA

Biotinylated cDNA








1 2





1

2

TAG-1

TAG-2



o Gene expression



o miRNA analysis


applications




ANNEAL

Ligate

PCR

TAG1

TAG2

cDNA or

gDNAPCR

HYBRIDIZE

amp

LABEL

dA

dG dT

dC -

ANNEAL

amp

Extend

TAG1

TAG2

HYBRIDIZE

amp

LABEL

gDNAPCR

SNP typing CF

CYP 450 RVP


CNV etc

RVP FAST GPP FAST

HYBRIDIZE

amp

LABEL

TAG1

TAG2 ANNEAL

amp

PCR

gDNA


30



2 hours



30 minutes



30 minutes





30 minutes







methods







TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

AAAAAAA RNA

AAAAAAA RNA

AAAAAAA RNA






TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

AAAAAAA RNA

AAAAAAA RNA

AAAAAAA RNA

cDNA

cDNA

cDNA




TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

cDNA

cDNA

cDNA




LMA Probe Design



Upstream LMA Probe

cDNA


Universal Primer 1

5rsquo 3rsquo












TAG

SAPE

Detection



lt3 Hours Setup











ANNEAL

Ligate

PCR

TAG1

TAG2

cDNA or

gDNAPCR

HYBRIDIZE

amp

LABEL





Upstream LMA Probes

T

Universal Primer 1

5rsquo

A

5rsquo

G



SNP

T



T

T


PCR Amplification

UP 1

P

No Amplification

B-UP 2



Universal Prime mix

UP 1

P

B-UP 2

UP 1

P

B-UP 2

Ligate

T T







TAG

SAPE

Detection



Bead Hyb 80 minutes







Incubation








Targeted PCR




Upstream LDR Probes

A T

G



SNP

A

A



A


Multiple

Ligation

cycles









TAG

SAPE

Detection






Forward primers

gDNA


5rsquo

3rsquo


A

T

5rsquo

3rsquo

G





Targeted PCR



A

5rsquo

3rsquo

G

5rsquo

3rsquo

T




A

5rsquo

3rsquo

G 5rsquo

3rsquo

T








xTAG


Detection

SAPE










xTAG primer

gDNA


Biotinylated primer

5rsquo 3rsquo

3rsquo

dATP dTTP dGTP dCTP

= Carbon Spacer











Detection

Sequence Detection

TAG


















Workflow

75



2 hours



30 minutes



30 minutes





30 minutes

76






0

500

1000

1500

2000

2500

3000

3500

4000


Sample

Sig

nal In

ten

sit

y (

MF

I)

let-7c

Let-7a

77


miR-107 vs miR-103

0

1000

2000

3000

4000

5000

6000


Sample

Sig

na

l In

ten

sit

y (

MF

I)

miR-103

miR-107









0

1000

2000

3000

4000

5000

6000

7000

8000

9000

10000

hsa-m

iR-1

07

hsa-m

iR-1

47

hsa-m

iR-1

81a

hsa-m

iR-1

92

hsa-m

iR-1

9b

hsa-m

iR-2

20a

hsa-m

iR-2

1hsa-m

iR-2

18

hsa-le

t-7c

hsa-m

iR-1

00

hsa-m

iR-1

25b

hsa-m

iR-

hsa-m

iR-2

05

hsa-m

iR-2

21

hsa-m

iR-2

00a

hsa-m

iR-2

00c

hsa-m

iR-2

06

hsa-m

iR-1

0a

hsa-m

iR-1

6

hsa-m

iR-9

hsa-m

iR-4

24

hsa-m

iR-2

0a

hsa-m

iR-2

0b

hsa-le

t-7a

hsa-m

iR-9

5hsa-m

iR-1

36

hsa-m

iR-1

95

hsa-m

iR-2

10

hsa-m

iR-2

12

hsa-m

iR-

hsa-m

iR-1

7

hsa-m

iR-1

22

hsa-m

iR-1

43

hsa-m

iR-1

45

hsa-m

iR-1

06a

hsa-m

iR-

hsa-m

iR-1

0b

hsa-m

iR-1

hsa-m

iR-2

23

hsa-m

iR-1

81b

hsa-m

iR-1

55

hsa-le

t-7g

hsa-le

t-7i

hsa-m

iR-9

6

hsa-m

iR-1

37

hsa-m

iR-1

85

hsa-m

iR-1

38

hsa-m

iR-1

98

hsa-m

iR-1

33a

hsa-m

iR-1

33b

hsa-m

iR-1

5a

hsa-m

iR-3

4a

hsa-m

iR-2

9a

hsa-m

iR-2

9b

hsa-m

iR-1

03

hsa-m

iR-2

22

hsa-m

iR-1

46a

cel-m

iR-3

9cel-m

iR-5

4

cel-m

iR-2

38

Regions

Ne

t S

ign

al In

ten

sit

y (

MF

I)

106a

17


Total RNA Samples

53-Plex Assay

miR-9 miR-122 miR-1

0

5000

10000

15000

20000

25000

Samples

Ne

t S

ign

al In

ten

sit

y (

MF

I)

miR-9

miR-122

miR-1






289704

12431797

4050

7044

12899

18030

24973

23884

21852

19249

11326

6573

3852

2471

0

5000

10000

15000

20000

25000

30000


Input Amount

Ne

t S

ign

al In

ten

sit

y (

MF

I)

200 amol


81


0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000



Ne

t S

ign

al in

ten

sit

y (

MF

I)

82

miR-133a

000

020

040

060

080

100

120


Total RNA Samples

Rela

tive C

han

ge t

o H

eart

FlexmiR v2

qRT-PCR

Tissue

Re

lati

ve

Ch

an

ge


LNP-miRNA

qRT-PCR

83

miRNA

Re

lati

ve

Ch

an

ge



0

5

10

15

20

25


miRNA

Rela

tive C

han

ge

FlexmiR v2

qRT-PCR

LNP-miRNA

qPCR

84



0

5000

10000

15000

20000

25000

30000

hsa-m

iR-1

07

hsa-m

iR-1

47

hsa-m

iR-1

81a

hsa-m

iR-1

92

hsa-m

iR-1

9b

hsa-m

iR-2

20a

hsa-m

iR-2

1hsa-m

iR-2

18

hsa-le

t-7c

hsa-m

iR-1

00

hsa-m

iR-1

25b

hsa-m

iR-

hsa-m

iR-2

05

hsa-m

iR-2

21

hsa-m

iR-2

00a

hsa-m

iR-2

00c

hsa-m

iR-2

06

hsa-m

iR-1

0a

hsa-m

iR-1

6hsa-m

iR-9

hsa-m

iR-4

24

hsa-m

iR-2

0a

hsa-m

iR-2

0b

hsa-le

t-7a

hsa-m

iR-9

5hsa-m

iR-1

36

hsa-m

iR-1

95

hsa-m

iR-2

10

hsa-m

iR-2

12

hsa-m

iR-

hsa-m

iR-1

7hsa-m

iR-1

22

hsa-m

iR-1

43

hsa-m

iR-1

45

hsa-m

ir-1

06a

hsa-m

iR-

hsa-m

iR-1

0b

hsa-m

iR-1

hsa-m

iR-2

23

hsa-m

iR-1

81b

hsa-le

t-155

hsa-le

t-7g

hsa-m

iR-7

ihsa-m

iR-9

6hsa-m

iR-1

37

hsa-m

iR-1

85

hsa-m

iR-1

38

hsa-m

iR-1

33a

hsa-m

iR-1

33b

hsa-m

iR-1

5a

hsa-m

iR-3

4a

hsa-m

iR-2

9a

hsa-m

iR-2

9b

hsa-m

iR-1

03

hsa-m

iR-2

22

hsa-m

iR-1

46a

hsa-m

iR-3

9cel-m

iR-3

9cel-m

iR-2

38

Regions

Ne

t S

ign

al in

ten

sit

y (

MF

I)

50ng

100ng

250ng

500ng








bull Flexible





Proteomic Assays



Nucleic Acids








or IgM





Multiplex Detection

Detection


IgG

1 2 3 4





















Y Y

Y

IgG1 IgG2

IgG3

IgG4 IgA



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Detection



Antigen

1 2 3








Y Y Y

Y

Y

Y Y

Y Y

Y Y

Y



Y


Detection

Multiplex Detection


















compounds





compounds



SAPE SAPE














SAPE SAPE


Detection

Multiplex Detection












543-550






GTPases 827 271-282














Single Plex Assays

To Luminex

Multiplex

Assays



Arrays

And

NGS

RT-PCR


Discovery

amp

Validation-1

An

aly

tes

S

am

ple

Samples Day

10s 100s 1000s 10000s

10

100

1000

10000 3D Throughput



6 x 384 platesday

2304 samplesday

1152000 dpday


MAGPIX

50 Plex

LX200

80-100 Plex

FLEXMAP 3D

500 Plex




concentrations

Quantify binding

events

Interrogate bead

with red laser

(635 nm)

Interrogate label

with green laser

(525 nm)

Sheath Fluid



green LED (525 nm)


LED (635 nm)


CCD imager


Capture


Genomic Assays










Detection

SAPE






HPV (high-risk

mucosal anogenital)




Quint et al 2009



HPV (high-risk

mucosal anogenital)



HPV (mucosal

anogenital)










Detection

RNA

Biotinylated cDNA








1 2





1

2

TAG-1

TAG-2



o Gene expression



o miRNA analysis


applications




ANNEAL

Ligate

PCR

TAG1

TAG2

cDNA or

gDNAPCR

HYBRIDIZE

amp

LABEL

dA

dG dT

dC -

ANNEAL

amp

Extend

TAG1

TAG2

HYBRIDIZE

amp

LABEL

gDNAPCR

SNP typing CF

CYP 450 RVP


CNV etc

RVP FAST GPP FAST

HYBRIDIZE

amp

LABEL

TAG1

TAG2 ANNEAL

amp

PCR

gDNA


30



2 hours



30 minutes



30 minutes





30 minutes







methods







TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

AAAAAAA RNA

AAAAAAA RNA

AAAAAAA RNA






TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

AAAAAAA RNA

AAAAAAA RNA

AAAAAAA RNA

cDNA

cDNA

cDNA




TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

cDNA

cDNA

cDNA




LMA Probe Design



Upstream LMA Probe

cDNA


Universal Primer 1

5rsquo 3rsquo












TAG

SAPE

Detection



lt3 Hours Setup











ANNEAL

Ligate

PCR

TAG1

TAG2

cDNA or

gDNAPCR

HYBRIDIZE

amp

LABEL





Upstream LMA Probes

T

Universal Primer 1

5rsquo

A

5rsquo

G



SNP

T



T

T


PCR Amplification

UP 1

P

No Amplification

B-UP 2



Universal Prime mix

UP 1

P

B-UP 2

UP 1

P

B-UP 2

Ligate

T T







TAG

SAPE

Detection



Bead Hyb 80 minutes







Incubation








Targeted PCR




Upstream LDR Probes

A T

G



SNP

A

A



A


Multiple

Ligation

cycles









TAG

SAPE

Detection






Forward primers

gDNA


5rsquo

3rsquo


A

T

5rsquo

3rsquo

G





Targeted PCR



A

5rsquo

3rsquo

G

5rsquo

3rsquo

T




A

5rsquo

3rsquo

G 5rsquo

3rsquo

T








xTAG


Detection

SAPE










xTAG primer

gDNA


Biotinylated primer

5rsquo 3rsquo

3rsquo

dATP dTTP dGTP dCTP

= Carbon Spacer











Detection

Sequence Detection

TAG


















Workflow

75



2 hours



30 minutes



30 minutes





30 minutes

76






0

500

1000

1500

2000

2500

3000

3500

4000


Sample

Sig

nal In

ten

sit

y (

MF

I)

let-7c

Let-7a

77


miR-107 vs miR-103

0

1000

2000

3000

4000

5000

6000


Sample

Sig

na

l In

ten

sit

y (

MF

I)

miR-103

miR-107









0

1000

2000

3000

4000

5000

6000

7000

8000

9000

10000

hsa-m

iR-1

07

hsa-m

iR-1

47

hsa-m

iR-1

81a

hsa-m

iR-1

92

hsa-m

iR-1

9b

hsa-m

iR-2

20a

hsa-m

iR-2

1hsa-m

iR-2

18

hsa-le

t-7c

hsa-m

iR-1

00

hsa-m

iR-1

25b

hsa-m

iR-

hsa-m

iR-2

05

hsa-m

iR-2

21

hsa-m

iR-2

00a

hsa-m

iR-2

00c

hsa-m

iR-2

06

hsa-m

iR-1

0a

hsa-m

iR-1

6

hsa-m

iR-9

hsa-m

iR-4

24

hsa-m

iR-2

0a

hsa-m

iR-2

0b

hsa-le

t-7a

hsa-m

iR-9

5hsa-m

iR-1

36

hsa-m

iR-1

95

hsa-m

iR-2

10

hsa-m

iR-2

12

hsa-m

iR-

hsa-m

iR-1

7

hsa-m

iR-1

22

hsa-m

iR-1

43

hsa-m

iR-1

45

hsa-m

iR-1

06a

hsa-m

iR-

hsa-m

iR-1

0b

hsa-m

iR-1

hsa-m

iR-2

23

hsa-m

iR-1

81b

hsa-m

iR-1

55

hsa-le

t-7g

hsa-le

t-7i

hsa-m

iR-9

6

hsa-m

iR-1

37

hsa-m

iR-1

85

hsa-m

iR-1

38

hsa-m

iR-1

98

hsa-m

iR-1

33a

hsa-m

iR-1

33b

hsa-m

iR-1

5a

hsa-m

iR-3

4a

hsa-m

iR-2

9a

hsa-m

iR-2

9b

hsa-m

iR-1

03

hsa-m

iR-2

22

hsa-m

iR-1

46a

cel-m

iR-3

9cel-m

iR-5

4

cel-m

iR-2

38

Regions

Ne

t S

ign

al In

ten

sit

y (

MF

I)

106a

17


Total RNA Samples

53-Plex Assay

miR-9 miR-122 miR-1

0

5000

10000

15000

20000

25000

Samples

Ne

t S

ign

al In

ten

sit

y (

MF

I)

miR-9

miR-122

miR-1






289704

12431797

4050

7044

12899

18030

24973

23884

21852

19249

11326

6573

3852

2471

0

5000

10000

15000

20000

25000

30000


Input Amount

Ne

t S

ign

al In

ten

sit

y (

MF

I)

200 amol


81


0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000



Ne

t S

ign

al in

ten

sit

y (

MF

I)

82

miR-133a

000

020

040

060

080

100

120


Total RNA Samples

Rela

tive C

han

ge t

o H

eart

FlexmiR v2

qRT-PCR

Tissue

Re

lati

ve

Ch

an

ge


LNP-miRNA

qRT-PCR

83

miRNA

Re

lati

ve

Ch

an

ge



0

5

10

15

20

25


miRNA

Rela

tive C

han

ge

FlexmiR v2

qRT-PCR

LNP-miRNA

qPCR

84



0

5000

10000

15000

20000

25000

30000

hsa-m

iR-1

07

hsa-m

iR-1

47

hsa-m

iR-1

81a

hsa-m

iR-1

92

hsa-m

iR-1

9b

hsa-m

iR-2

20a

hsa-m

iR-2

1hsa-m

iR-2

18

hsa-le

t-7c

hsa-m

iR-1

00

hsa-m

iR-1

25b

hsa-m

iR-

hsa-m

iR-2

05

hsa-m

iR-2

21

hsa-m

iR-2

00a

hsa-m

iR-2

00c

hsa-m

iR-2

06

hsa-m

iR-1

0a

hsa-m

iR-1

6hsa-m

iR-9

hsa-m

iR-4

24

hsa-m

iR-2

0a

hsa-m

iR-2

0b

hsa-le

t-7a

hsa-m

iR-9

5hsa-m

iR-1

36

hsa-m

iR-1

95

hsa-m

iR-2

10

hsa-m

iR-2

12

hsa-m

iR-

hsa-m

iR-1

7hsa-m

iR-1

22

hsa-m

iR-1

43

hsa-m

iR-1

45

hsa-m

ir-1

06a

hsa-m

iR-

hsa-m

iR-1

0b

hsa-m

iR-1

hsa-m

iR-2

23

hsa-m

iR-1

81b

hsa-le

t-155

hsa-le

t-7g

hsa-m

iR-7

ihsa-m

iR-9

6hsa-m

iR-1

37

hsa-m

iR-1

85

hsa-m

iR-1

38

hsa-m

iR-1

33a

hsa-m

iR-1

33b

hsa-m

iR-1

5a

hsa-m

iR-3

4a

hsa-m

iR-2

9a

hsa-m

iR-2

9b

hsa-m

iR-1

03

hsa-m

iR-2

22

hsa-m

iR-1

46a

hsa-m

iR-3

9cel-m

iR-3

9cel-m

iR-2

38

Regions

Ne

t S

ign

al in

ten

sit

y (

MF

I)

50ng

100ng

250ng

500ng








bull Flexible





Proteomic Assays



Nucleic Acids








or IgM





Multiplex Detection

Detection


IgG

1 2 3 4





















Y Y

Y

IgG1 IgG2

IgG3

IgG4 IgA



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Detection



Antigen

1 2 3








Y Y Y

Y

Y

Y Y

Y Y

Y Y

Y



Y


Detection

Multiplex Detection


















compounds





compounds



SAPE SAPE














SAPE SAPE


Detection

Multiplex Detection












543-550






GTPases 827 271-282














Single Plex Assays

To Luminex

Multiplex

Assays






concentrations

Quantify binding

events

Interrogate bead

with red laser

(635 nm)

Interrogate label

with green laser

(525 nm)

Sheath Fluid



green LED (525 nm)


LED (635 nm)


CCD imager


Capture


Genomic Assays










Detection

SAPE






HPV (high-risk

mucosal anogenital)




Quint et al 2009



HPV (high-risk

mucosal anogenital)



HPV (mucosal

anogenital)










Detection

RNA

Biotinylated cDNA








1 2





1

2

TAG-1

TAG-2



o Gene expression



o miRNA analysis


applications




ANNEAL

Ligate

PCR

TAG1

TAG2

cDNA or

gDNAPCR

HYBRIDIZE

amp

LABEL

dA

dG dT

dC -

ANNEAL

amp

Extend

TAG1

TAG2

HYBRIDIZE

amp

LABEL

gDNAPCR

SNP typing CF

CYP 450 RVP


CNV etc

RVP FAST GPP FAST

HYBRIDIZE

amp

LABEL

TAG1

TAG2 ANNEAL

amp

PCR

gDNA


30



2 hours



30 minutes



30 minutes





30 minutes







methods







TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

AAAAAAA RNA

AAAAAAA RNA

AAAAAAA RNA






TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

AAAAAAA RNA

AAAAAAA RNA

AAAAAAA RNA

cDNA

cDNA

cDNA




TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

cDNA

cDNA

cDNA




LMA Probe Design



Upstream LMA Probe

cDNA


Universal Primer 1

5rsquo 3rsquo












TAG

SAPE

Detection



lt3 Hours Setup











ANNEAL

Ligate

PCR

TAG1

TAG2

cDNA or

gDNAPCR

HYBRIDIZE

amp

LABEL





Upstream LMA Probes

T

Universal Primer 1

5rsquo

A

5rsquo

G



SNP

T



T

T


PCR Amplification

UP 1

P

No Amplification

B-UP 2



Universal Prime mix

UP 1

P

B-UP 2

UP 1

P

B-UP 2

Ligate

T T







TAG

SAPE

Detection



Bead Hyb 80 minutes







Incubation








Targeted PCR




Upstream LDR Probes

A T

G



SNP

A

A



A


Multiple

Ligation

cycles









TAG

SAPE

Detection






Forward primers

gDNA


5rsquo

3rsquo


A

T

5rsquo

3rsquo

G





Targeted PCR



A

5rsquo

3rsquo

G

5rsquo

3rsquo

T




A

5rsquo

3rsquo

G 5rsquo

3rsquo

T








xTAG


Detection

SAPE










xTAG primer

gDNA


Biotinylated primer

5rsquo 3rsquo

3rsquo

dATP dTTP dGTP dCTP

= Carbon Spacer











Detection

Sequence Detection

TAG


















Workflow

75



2 hours



30 minutes



30 minutes





30 minutes

76






0

500

1000

1500

2000

2500

3000

3500

4000


Sample

Sig

nal In

ten

sit

y (

MF

I)

let-7c

Let-7a

77


miR-107 vs miR-103

0

1000

2000

3000

4000

5000

6000


Sample

Sig

na

l In

ten

sit

y (

MF

I)

miR-103

miR-107









0

1000

2000

3000

4000

5000

6000

7000

8000

9000

10000

hsa-m

iR-1

07

hsa-m

iR-1

47

hsa-m

iR-1

81a

hsa-m

iR-1

92

hsa-m

iR-1

9b

hsa-m

iR-2

20a

hsa-m

iR-2

1hsa-m

iR-2

18

hsa-le

t-7c

hsa-m

iR-1

00

hsa-m

iR-1

25b

hsa-m

iR-

hsa-m

iR-2

05

hsa-m

iR-2

21

hsa-m

iR-2

00a

hsa-m

iR-2

00c

hsa-m

iR-2

06

hsa-m

iR-1

0a

hsa-m

iR-1

6

hsa-m

iR-9

hsa-m

iR-4

24

hsa-m

iR-2

0a

hsa-m

iR-2

0b

hsa-le

t-7a

hsa-m

iR-9

5hsa-m

iR-1

36

hsa-m

iR-1

95

hsa-m

iR-2

10

hsa-m

iR-2

12

hsa-m

iR-

hsa-m

iR-1

7

hsa-m

iR-1

22

hsa-m

iR-1

43

hsa-m

iR-1

45

hsa-m

iR-1

06a

hsa-m

iR-

hsa-m

iR-1

0b

hsa-m

iR-1

hsa-m

iR-2

23

hsa-m

iR-1

81b

hsa-m

iR-1

55

hsa-le

t-7g

hsa-le

t-7i

hsa-m

iR-9

6

hsa-m

iR-1

37

hsa-m

iR-1

85

hsa-m

iR-1

38

hsa-m

iR-1

98

hsa-m

iR-1

33a

hsa-m

iR-1

33b

hsa-m

iR-1

5a

hsa-m

iR-3

4a

hsa-m

iR-2

9a

hsa-m

iR-2

9b

hsa-m

iR-1

03

hsa-m

iR-2

22

hsa-m

iR-1

46a

cel-m

iR-3

9cel-m

iR-5

4

cel-m

iR-2

38

Regions

Ne

t S

ign

al In

ten

sit

y (

MF

I)

106a

17


Total RNA Samples

53-Plex Assay

miR-9 miR-122 miR-1

0

5000

10000

15000

20000

25000

Samples

Ne

t S

ign

al In

ten

sit

y (

MF

I)

miR-9

miR-122

miR-1






289704

12431797

4050

7044

12899

18030

24973

23884

21852

19249

11326

6573

3852

2471

0

5000

10000

15000

20000

25000

30000


Input Amount

Ne

t S

ign

al In

ten

sit

y (

MF

I)

200 amol


81


0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000



Ne

t S

ign

al in

ten

sit

y (

MF

I)

82

miR-133a

000

020

040

060

080

100

120


Total RNA Samples

Rela

tive C

han

ge t

o H

eart

FlexmiR v2

qRT-PCR

Tissue

Re

lati

ve

Ch

an

ge


LNP-miRNA

qRT-PCR

83

miRNA

Re

lati

ve

Ch

an

ge



0

5

10

15

20

25


miRNA

Rela

tive C

han

ge

FlexmiR v2

qRT-PCR

LNP-miRNA

qPCR

84



0

5000

10000

15000

20000

25000

30000

hsa-m

iR-1

07

hsa-m

iR-1

47

hsa-m

iR-1

81a

hsa-m

iR-1

92

hsa-m

iR-1

9b

hsa-m

iR-2

20a

hsa-m

iR-2

1hsa-m

iR-2

18

hsa-le

t-7c

hsa-m

iR-1

00

hsa-m

iR-1

25b

hsa-m

iR-

hsa-m

iR-2

05

hsa-m

iR-2

21

hsa-m

iR-2

00a

hsa-m

iR-2

00c

hsa-m

iR-2

06

hsa-m

iR-1

0a

hsa-m

iR-1

6hsa-m

iR-9

hsa-m

iR-4

24

hsa-m

iR-2

0a

hsa-m

iR-2

0b

hsa-le

t-7a

hsa-m

iR-9

5hsa-m

iR-1

36

hsa-m

iR-1

95

hsa-m

iR-2

10

hsa-m

iR-2

12

hsa-m

iR-

hsa-m

iR-1

7hsa-m

iR-1

22

hsa-m

iR-1

43

hsa-m

iR-1

45

hsa-m

ir-1

06a

hsa-m

iR-

hsa-m

iR-1

0b

hsa-m

iR-1

hsa-m

iR-2

23

hsa-m

iR-1

81b

hsa-le

t-155

hsa-le

t-7g

hsa-m

iR-7

ihsa-m

iR-9

6hsa-m

iR-1

37

hsa-m

iR-1

85

hsa-m

iR-1

38

hsa-m

iR-1

33a

hsa-m

iR-1

33b

hsa-m

iR-1

5a

hsa-m

iR-3

4a

hsa-m

iR-2

9a

hsa-m

iR-2

9b

hsa-m

iR-1

03

hsa-m

iR-2

22

hsa-m

iR-1

46a

hsa-m

iR-3

9cel-m

iR-3

9cel-m

iR-2

38

Regions

Ne

t S

ign

al in

ten

sit

y (

MF

I)

50ng

100ng

250ng

500ng








bull Flexible





Proteomic Assays



Nucleic Acids








or IgM





Multiplex Detection

Detection


IgG

1 2 3 4





















Y Y

Y

IgG1 IgG2

IgG3

IgG4 IgA



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Detection



Antigen

1 2 3








Y Y Y

Y

Y

Y Y

Y Y

Y Y

Y



Y


Detection

Multiplex Detection


















compounds





compounds



SAPE SAPE














SAPE SAPE


Detection

Multiplex Detection












543-550






GTPases 827 271-282














Single Plex Assays

To Luminex

Multiplex

Assays



Genomic Assays










Detection

SAPE






HPV (high-risk

mucosal anogenital)




Quint et al 2009



HPV (high-risk

mucosal anogenital)



HPV (mucosal

anogenital)










Detection

RNA

Biotinylated cDNA








1 2





1

2

TAG-1

TAG-2



o Gene expression



o miRNA analysis


applications




ANNEAL

Ligate

PCR

TAG1

TAG2

cDNA or

gDNAPCR

HYBRIDIZE

amp

LABEL

dA

dG dT

dC -

ANNEAL

amp

Extend

TAG1

TAG2

HYBRIDIZE

amp

LABEL

gDNAPCR

SNP typing CF

CYP 450 RVP


CNV etc

RVP FAST GPP FAST

HYBRIDIZE

amp

LABEL

TAG1

TAG2 ANNEAL

amp

PCR

gDNA


30



2 hours



30 minutes



30 minutes





30 minutes







methods







TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

AAAAAAA RNA

AAAAAAA RNA

AAAAAAA RNA






TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

AAAAAAA RNA

AAAAAAA RNA

AAAAAAA RNA

cDNA

cDNA

cDNA




TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

cDNA

cDNA

cDNA




LMA Probe Design



Upstream LMA Probe

cDNA


Universal Primer 1

5rsquo 3rsquo












TAG

SAPE

Detection



lt3 Hours Setup











ANNEAL

Ligate

PCR

TAG1

TAG2

cDNA or

gDNAPCR

HYBRIDIZE

amp

LABEL





Upstream LMA Probes

T

Universal Primer 1

5rsquo

A

5rsquo

G



SNP

T



T

T


PCR Amplification

UP 1

P

No Amplification

B-UP 2



Universal Prime mix

UP 1

P

B-UP 2

UP 1

P

B-UP 2

Ligate

T T







TAG

SAPE

Detection



Bead Hyb 80 minutes







Incubation








Targeted PCR




Upstream LDR Probes

A T

G



SNP

A

A



A


Multiple

Ligation

cycles









TAG

SAPE

Detection






Forward primers

gDNA


5rsquo

3rsquo


A

T

5rsquo

3rsquo

G





Targeted PCR



A

5rsquo

3rsquo

G

5rsquo

3rsquo

T




A

5rsquo

3rsquo

G 5rsquo

3rsquo

T








xTAG


Detection

SAPE










xTAG primer

gDNA


Biotinylated primer

5rsquo 3rsquo

3rsquo

dATP dTTP dGTP dCTP

= Carbon Spacer











Detection

Sequence Detection

TAG


















Workflow

75



2 hours



30 minutes



30 minutes





30 minutes

76






0

500

1000

1500

2000

2500

3000

3500

4000


Sample

Sig

nal In

ten

sit

y (

MF

I)

let-7c

Let-7a

77


miR-107 vs miR-103

0

1000

2000

3000

4000

5000

6000


Sample

Sig

na

l In

ten

sit

y (

MF

I)

miR-103

miR-107









0

1000

2000

3000

4000

5000

6000

7000

8000

9000

10000

hsa-m

iR-1

07

hsa-m

iR-1

47

hsa-m

iR-1

81a

hsa-m

iR-1

92

hsa-m

iR-1

9b

hsa-m

iR-2

20a

hsa-m

iR-2

1hsa-m

iR-2

18

hsa-le

t-7c

hsa-m

iR-1

00

hsa-m

iR-1

25b

hsa-m

iR-

hsa-m

iR-2

05

hsa-m

iR-2

21

hsa-m

iR-2

00a

hsa-m

iR-2

00c

hsa-m

iR-2

06

hsa-m

iR-1

0a

hsa-m

iR-1

6

hsa-m

iR-9

hsa-m

iR-4

24

hsa-m

iR-2

0a

hsa-m

iR-2

0b

hsa-le

t-7a

hsa-m

iR-9

5hsa-m

iR-1

36

hsa-m

iR-1

95

hsa-m

iR-2

10

hsa-m

iR-2

12

hsa-m

iR-

hsa-m

iR-1

7

hsa-m

iR-1

22

hsa-m

iR-1

43

hsa-m

iR-1

45

hsa-m

iR-1

06a

hsa-m

iR-

hsa-m

iR-1

0b

hsa-m

iR-1

hsa-m

iR-2

23

hsa-m

iR-1

81b

hsa-m

iR-1

55

hsa-le

t-7g

hsa-le

t-7i

hsa-m

iR-9

6

hsa-m

iR-1

37

hsa-m

iR-1

85

hsa-m

iR-1

38

hsa-m

iR-1

98

hsa-m

iR-1

33a

hsa-m

iR-1

33b

hsa-m

iR-1

5a

hsa-m

iR-3

4a

hsa-m

iR-2

9a

hsa-m

iR-2

9b

hsa-m

iR-1

03

hsa-m

iR-2

22

hsa-m

iR-1

46a

cel-m

iR-3

9cel-m

iR-5

4

cel-m

iR-2

38

Regions

Ne

t S

ign

al In

ten

sit

y (

MF

I)

106a

17


Total RNA Samples

53-Plex Assay

miR-9 miR-122 miR-1

0

5000

10000

15000

20000

25000

Samples

Ne

t S

ign

al In

ten

sit

y (

MF

I)

miR-9

miR-122

miR-1






289704

12431797

4050

7044

12899

18030

24973

23884

21852

19249

11326

6573

3852

2471

0

5000

10000

15000

20000

25000

30000


Input Amount

Ne

t S

ign

al In

ten

sit

y (

MF

I)

200 amol


81


0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000



Ne

t S

ign

al in

ten

sit

y (

MF

I)

82

miR-133a

000

020

040

060

080

100

120


Total RNA Samples

Rela

tive C

han

ge t

o H

eart

FlexmiR v2

qRT-PCR

Tissue

Re

lati

ve

Ch

an

ge


LNP-miRNA

qRT-PCR

83

miRNA

Re

lati

ve

Ch

an

ge



0

5

10

15

20

25


miRNA

Rela

tive C

han

ge

FlexmiR v2

qRT-PCR

LNP-miRNA

qPCR

84



0

5000

10000

15000

20000

25000

30000

hsa-m

iR-1

07

hsa-m

iR-1

47

hsa-m

iR-1

81a

hsa-m

iR-1

92

hsa-m

iR-1

9b

hsa-m

iR-2

20a

hsa-m

iR-2

1hsa-m

iR-2

18

hsa-le

t-7c

hsa-m

iR-1

00

hsa-m

iR-1

25b

hsa-m

iR-

hsa-m

iR-2

05

hsa-m

iR-2

21

hsa-m

iR-2

00a

hsa-m

iR-2

00c

hsa-m

iR-2

06

hsa-m

iR-1

0a

hsa-m

iR-1

6hsa-m

iR-9

hsa-m

iR-4

24

hsa-m

iR-2

0a

hsa-m

iR-2

0b

hsa-le

t-7a

hsa-m

iR-9

5hsa-m

iR-1

36

hsa-m

iR-1

95

hsa-m

iR-2

10

hsa-m

iR-2

12

hsa-m

iR-

hsa-m

iR-1

7hsa-m

iR-1

22

hsa-m

iR-1

43

hsa-m

iR-1

45

hsa-m

ir-1

06a

hsa-m

iR-

hsa-m

iR-1

0b

hsa-m

iR-1

hsa-m

iR-2

23

hsa-m

iR-1

81b

hsa-le

t-155

hsa-le

t-7g

hsa-m

iR-7

ihsa-m

iR-9

6hsa-m

iR-1

37

hsa-m

iR-1

85

hsa-m

iR-1

38

hsa-m

iR-1

33a

hsa-m

iR-1

33b

hsa-m

iR-1

5a

hsa-m

iR-3

4a

hsa-m

iR-2

9a

hsa-m

iR-2

9b

hsa-m

iR-1

03

hsa-m

iR-2

22

hsa-m

iR-1

46a

hsa-m

iR-3

9cel-m

iR-3

9cel-m

iR-2

38

Regions

Ne

t S

ign

al in

ten

sit

y (

MF

I)

50ng

100ng

250ng

500ng








bull Flexible





Proteomic Assays



Nucleic Acids








or IgM





Multiplex Detection

Detection


IgG

1 2 3 4





















Y Y

Y

IgG1 IgG2

IgG3

IgG4 IgA



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Detection



Antigen

1 2 3








Y Y Y

Y

Y

Y Y

Y Y

Y Y

Y



Y


Detection

Multiplex Detection


















compounds





compounds



SAPE SAPE














SAPE SAPE


Detection

Multiplex Detection












543-550






GTPases 827 271-282














Single Plex Assays

To Luminex

Multiplex

Assays












Detection

SAPE






HPV (high-risk

mucosal anogenital)




Quint et al 2009



HPV (high-risk

mucosal anogenital)



HPV (mucosal

anogenital)










Detection

RNA

Biotinylated cDNA








1 2





1

2

TAG-1

TAG-2



o Gene expression



o miRNA analysis


applications




ANNEAL

Ligate

PCR

TAG1

TAG2

cDNA or

gDNAPCR

HYBRIDIZE

amp

LABEL

dA

dG dT

dC -

ANNEAL

amp

Extend

TAG1

TAG2

HYBRIDIZE

amp

LABEL

gDNAPCR

SNP typing CF

CYP 450 RVP


CNV etc

RVP FAST GPP FAST

HYBRIDIZE

amp

LABEL

TAG1

TAG2 ANNEAL

amp

PCR

gDNA


30



2 hours



30 minutes



30 minutes





30 minutes







methods







TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

AAAAAAA RNA

AAAAAAA RNA

AAAAAAA RNA






TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

AAAAAAA RNA

AAAAAAA RNA

AAAAAAA RNA

cDNA

cDNA

cDNA




TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

cDNA

cDNA

cDNA




LMA Probe Design



Upstream LMA Probe

cDNA


Universal Primer 1

5rsquo 3rsquo












TAG

SAPE

Detection



lt3 Hours Setup











ANNEAL

Ligate

PCR

TAG1

TAG2

cDNA or

gDNAPCR

HYBRIDIZE

amp

LABEL





Upstream LMA Probes

T

Universal Primer 1

5rsquo

A

5rsquo

G



SNP

T



T

T


PCR Amplification

UP 1

P

No Amplification

B-UP 2



Universal Prime mix

UP 1

P

B-UP 2

UP 1

P

B-UP 2

Ligate

T T







TAG

SAPE

Detection



Bead Hyb 80 minutes







Incubation








Targeted PCR




Upstream LDR Probes

A T

G



SNP

A

A



A


Multiple

Ligation

cycles









TAG

SAPE

Detection






Forward primers

gDNA


5rsquo

3rsquo


A

T

5rsquo

3rsquo

G





Targeted PCR



A

5rsquo

3rsquo

G

5rsquo

3rsquo

T




A

5rsquo

3rsquo

G 5rsquo

3rsquo

T








xTAG


Detection

SAPE










xTAG primer

gDNA


Biotinylated primer

5rsquo 3rsquo

3rsquo

dATP dTTP dGTP dCTP

= Carbon Spacer











Detection

Sequence Detection

TAG


















Workflow

75



2 hours



30 minutes



30 minutes





30 minutes

76






0

500

1000

1500

2000

2500

3000

3500

4000


Sample

Sig

nal In

ten

sit

y (

MF

I)

let-7c

Let-7a

77


miR-107 vs miR-103

0

1000

2000

3000

4000

5000

6000


Sample

Sig

na

l In

ten

sit

y (

MF

I)

miR-103

miR-107









0

1000

2000

3000

4000

5000

6000

7000

8000

9000

10000

hsa-m

iR-1

07

hsa-m

iR-1

47

hsa-m

iR-1

81a

hsa-m

iR-1

92

hsa-m

iR-1

9b

hsa-m

iR-2

20a

hsa-m

iR-2

1hsa-m

iR-2

18

hsa-le

t-7c

hsa-m

iR-1

00

hsa-m

iR-1

25b

hsa-m

iR-

hsa-m

iR-2

05

hsa-m

iR-2

21

hsa-m

iR-2

00a

hsa-m

iR-2

00c

hsa-m

iR-2

06

hsa-m

iR-1

0a

hsa-m

iR-1

6

hsa-m

iR-9

hsa-m

iR-4

24

hsa-m

iR-2

0a

hsa-m

iR-2

0b

hsa-le

t-7a

hsa-m

iR-9

5hsa-m

iR-1

36

hsa-m

iR-1

95

hsa-m

iR-2

10

hsa-m

iR-2

12

hsa-m

iR-

hsa-m

iR-1

7

hsa-m

iR-1

22

hsa-m

iR-1

43

hsa-m

iR-1

45

hsa-m

iR-1

06a

hsa-m

iR-

hsa-m

iR-1

0b

hsa-m

iR-1

hsa-m

iR-2

23

hsa-m

iR-1

81b

hsa-m

iR-1

55

hsa-le

t-7g

hsa-le

t-7i

hsa-m

iR-9

6

hsa-m

iR-1

37

hsa-m

iR-1

85

hsa-m

iR-1

38

hsa-m

iR-1

98

hsa-m

iR-1

33a

hsa-m

iR-1

33b

hsa-m

iR-1

5a

hsa-m

iR-3

4a

hsa-m

iR-2

9a

hsa-m

iR-2

9b

hsa-m

iR-1

03

hsa-m

iR-2

22

hsa-m

iR-1

46a

cel-m

iR-3

9cel-m

iR-5

4

cel-m

iR-2

38

Regions

Ne

t S

ign

al In

ten

sit

y (

MF

I)

106a

17


Total RNA Samples

53-Plex Assay

miR-9 miR-122 miR-1

0

5000

10000

15000

20000

25000

Samples

Ne

t S

ign

al In

ten

sit

y (

MF

I)

miR-9

miR-122

miR-1






289704

12431797

4050

7044

12899

18030

24973

23884

21852

19249

11326

6573

3852

2471

0

5000

10000

15000

20000

25000

30000


Input Amount

Ne

t S

ign

al In

ten

sit

y (

MF

I)

200 amol


81


0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000



Ne

t S

ign

al in

ten

sit

y (

MF

I)

82

miR-133a

000

020

040

060

080

100

120


Total RNA Samples

Rela

tive C

han

ge t

o H

eart

FlexmiR v2

qRT-PCR

Tissue

Re

lati

ve

Ch

an

ge


LNP-miRNA

qRT-PCR

83

miRNA

Re

lati

ve

Ch

an

ge



0

5

10

15

20

25


miRNA

Rela

tive C

han

ge

FlexmiR v2

qRT-PCR

LNP-miRNA

qPCR

84



0

5000

10000

15000

20000

25000

30000

hsa-m

iR-1

07

hsa-m

iR-1

47

hsa-m

iR-1

81a

hsa-m

iR-1

92

hsa-m

iR-1

9b

hsa-m

iR-2

20a

hsa-m

iR-2

1hsa-m

iR-2

18

hsa-le

t-7c

hsa-m

iR-1

00

hsa-m

iR-1

25b

hsa-m

iR-

hsa-m

iR-2

05

hsa-m

iR-2

21

hsa-m

iR-2

00a

hsa-m

iR-2

00c

hsa-m

iR-2

06

hsa-m

iR-1

0a

hsa-m

iR-1

6hsa-m

iR-9

hsa-m

iR-4

24

hsa-m

iR-2

0a

hsa-m

iR-2

0b

hsa-le

t-7a

hsa-m

iR-9

5hsa-m

iR-1

36

hsa-m

iR-1

95

hsa-m

iR-2

10

hsa-m

iR-2

12

hsa-m

iR-

hsa-m

iR-1

7hsa-m

iR-1

22

hsa-m

iR-1

43

hsa-m

iR-1

45

hsa-m

ir-1

06a

hsa-m

iR-

hsa-m

iR-1

0b

hsa-m

iR-1

hsa-m

iR-2

23

hsa-m

iR-1

81b

hsa-le

t-155

hsa-le

t-7g

hsa-m

iR-7

ihsa-m

iR-9

6hsa-m

iR-1

37

hsa-m

iR-1

85

hsa-m

iR-1

38

hsa-m

iR-1

33a

hsa-m

iR-1

33b

hsa-m

iR-1

5a

hsa-m

iR-3

4a

hsa-m

iR-2

9a

hsa-m

iR-2

9b

hsa-m

iR-1

03

hsa-m

iR-2

22

hsa-m

iR-1

46a

hsa-m

iR-3

9cel-m

iR-3

9cel-m

iR-2

38

Regions

Ne

t S

ign

al in

ten

sit

y (

MF

I)

50ng

100ng

250ng

500ng








bull Flexible





Proteomic Assays



Nucleic Acids








or IgM





Multiplex Detection

Detection


IgG

1 2 3 4





















Y Y

Y

IgG1 IgG2

IgG3

IgG4 IgA



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Detection



Antigen

1 2 3








Y Y Y

Y

Y

Y Y

Y Y

Y Y

Y



Y


Detection

Multiplex Detection


















compounds





compounds



SAPE SAPE














SAPE SAPE


Detection

Multiplex Detection












543-550






GTPases 827 271-282














Single Plex Assays

To Luminex

Multiplex

Assays











Detection

SAPE






HPV (high-risk

mucosal anogenital)




Quint et al 2009



HPV (high-risk

mucosal anogenital)



HPV (mucosal

anogenital)










Detection

RNA

Biotinylated cDNA








1 2





1

2

TAG-1

TAG-2



o Gene expression



o miRNA analysis


applications




ANNEAL

Ligate

PCR

TAG1

TAG2

cDNA or

gDNAPCR

HYBRIDIZE

amp

LABEL

dA

dG dT

dC -

ANNEAL

amp

Extend

TAG1

TAG2

HYBRIDIZE

amp

LABEL

gDNAPCR

SNP typing CF

CYP 450 RVP


CNV etc

RVP FAST GPP FAST

HYBRIDIZE

amp

LABEL

TAG1

TAG2 ANNEAL

amp

PCR

gDNA


30



2 hours



30 minutes



30 minutes





30 minutes







methods







TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

AAAAAAA RNA

AAAAAAA RNA

AAAAAAA RNA






TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

AAAAAAA RNA

AAAAAAA RNA

AAAAAAA RNA

cDNA

cDNA

cDNA




TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

cDNA

cDNA

cDNA




LMA Probe Design



Upstream LMA Probe

cDNA


Universal Primer 1

5rsquo 3rsquo












TAG

SAPE

Detection



lt3 Hours Setup











ANNEAL

Ligate

PCR

TAG1

TAG2

cDNA or

gDNAPCR

HYBRIDIZE

amp

LABEL





Upstream LMA Probes

T

Universal Primer 1

5rsquo

A

5rsquo

G



SNP

T



T

T


PCR Amplification

UP 1

P

No Amplification

B-UP 2



Universal Prime mix

UP 1

P

B-UP 2

UP 1

P

B-UP 2

Ligate

T T







TAG

SAPE

Detection



Bead Hyb 80 minutes







Incubation








Targeted PCR




Upstream LDR Probes

A T

G



SNP

A

A



A


Multiple

Ligation

cycles









TAG

SAPE

Detection






Forward primers

gDNA


5rsquo

3rsquo


A

T

5rsquo

3rsquo

G





Targeted PCR



A

5rsquo

3rsquo

G

5rsquo

3rsquo

T




A

5rsquo

3rsquo

G 5rsquo

3rsquo

T








xTAG


Detection

SAPE










xTAG primer

gDNA


Biotinylated primer

5rsquo 3rsquo

3rsquo

dATP dTTP dGTP dCTP

= Carbon Spacer











Detection

Sequence Detection

TAG


















Workflow

75



2 hours



30 minutes



30 minutes





30 minutes

76






0

500

1000

1500

2000

2500

3000

3500

4000


Sample

Sig

nal In

ten

sit

y (

MF

I)

let-7c

Let-7a

77


miR-107 vs miR-103

0

1000

2000

3000

4000

5000

6000


Sample

Sig

na

l In

ten

sit

y (

MF

I)

miR-103

miR-107









0

1000

2000

3000

4000

5000

6000

7000

8000

9000

10000

hsa-m

iR-1

07

hsa-m

iR-1

47

hsa-m

iR-1

81a

hsa-m

iR-1

92

hsa-m

iR-1

9b

hsa-m

iR-2

20a

hsa-m

iR-2

1hsa-m

iR-2

18

hsa-le

t-7c

hsa-m

iR-1

00

hsa-m

iR-1

25b

hsa-m

iR-

hsa-m

iR-2

05

hsa-m

iR-2

21

hsa-m

iR-2

00a

hsa-m

iR-2

00c

hsa-m

iR-2

06

hsa-m

iR-1

0a

hsa-m

iR-1

6

hsa-m

iR-9

hsa-m

iR-4

24

hsa-m

iR-2

0a

hsa-m

iR-2

0b

hsa-le

t-7a

hsa-m

iR-9

5hsa-m

iR-1

36

hsa-m

iR-1

95

hsa-m

iR-2

10

hsa-m

iR-2

12

hsa-m

iR-

hsa-m

iR-1

7

hsa-m

iR-1

22

hsa-m

iR-1

43

hsa-m

iR-1

45

hsa-m

iR-1

06a

hsa-m

iR-

hsa-m

iR-1

0b

hsa-m

iR-1

hsa-m

iR-2

23

hsa-m

iR-1

81b

hsa-m

iR-1

55

hsa-le

t-7g

hsa-le

t-7i

hsa-m

iR-9

6

hsa-m

iR-1

37

hsa-m

iR-1

85

hsa-m

iR-1

38

hsa-m

iR-1

98

hsa-m

iR-1

33a

hsa-m

iR-1

33b

hsa-m

iR-1

5a

hsa-m

iR-3

4a

hsa-m

iR-2

9a

hsa-m

iR-2

9b

hsa-m

iR-1

03

hsa-m

iR-2

22

hsa-m

iR-1

46a

cel-m

iR-3

9cel-m

iR-5

4

cel-m

iR-2

38

Regions

Ne

t S

ign

al In

ten

sit

y (

MF

I)

106a

17


Total RNA Samples

53-Plex Assay

miR-9 miR-122 miR-1

0

5000

10000

15000

20000

25000

Samples

Ne

t S

ign

al In

ten

sit

y (

MF

I)

miR-9

miR-122

miR-1






289704

12431797

4050

7044

12899

18030

24973

23884

21852

19249

11326

6573

3852

2471

0

5000

10000

15000

20000

25000

30000


Input Amount

Ne

t S

ign

al In

ten

sit

y (

MF

I)

200 amol


81


0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000



Ne

t S

ign

al in

ten

sit

y (

MF

I)

82

miR-133a

000

020

040

060

080

100

120


Total RNA Samples

Rela

tive C

han

ge t

o H

eart

FlexmiR v2

qRT-PCR

Tissue

Re

lati

ve

Ch

an

ge


LNP-miRNA

qRT-PCR

83

miRNA

Re

lati

ve

Ch

an

ge



0

5

10

15

20

25


miRNA

Rela

tive C

han

ge

FlexmiR v2

qRT-PCR

LNP-miRNA

qPCR

84



0

5000

10000

15000

20000

25000

30000

hsa-m

iR-1

07

hsa-m

iR-1

47

hsa-m

iR-1

81a

hsa-m

iR-1

92

hsa-m

iR-1

9b

hsa-m

iR-2

20a

hsa-m

iR-2

1hsa-m

iR-2

18

hsa-le

t-7c

hsa-m

iR-1

00

hsa-m

iR-1

25b

hsa-m

iR-

hsa-m

iR-2

05

hsa-m

iR-2

21

hsa-m

iR-2

00a

hsa-m

iR-2

00c

hsa-m

iR-2

06

hsa-m

iR-1

0a

hsa-m

iR-1

6hsa-m

iR-9

hsa-m

iR-4

24

hsa-m

iR-2

0a

hsa-m

iR-2

0b

hsa-le

t-7a

hsa-m

iR-9

5hsa-m

iR-1

36

hsa-m

iR-1

95

hsa-m

iR-2

10

hsa-m

iR-2

12

hsa-m

iR-

hsa-m

iR-1

7hsa-m

iR-1

22

hsa-m

iR-1

43

hsa-m

iR-1

45

hsa-m

ir-1

06a

hsa-m

iR-

hsa-m

iR-1

0b

hsa-m

iR-1

hsa-m

iR-2

23

hsa-m

iR-1

81b

hsa-le

t-155

hsa-le

t-7g

hsa-m

iR-7

ihsa-m

iR-9

6hsa-m

iR-1

37

hsa-m

iR-1

85

hsa-m

iR-1

38

hsa-m

iR-1

33a

hsa-m

iR-1

33b

hsa-m

iR-1

5a

hsa-m

iR-3

4a

hsa-m

iR-2

9a

hsa-m

iR-2

9b

hsa-m

iR-1

03

hsa-m

iR-2

22

hsa-m

iR-1

46a

hsa-m

iR-3

9cel-m

iR-3

9cel-m

iR-2

38

Regions

Ne

t S

ign

al in

ten

sit

y (

MF

I)

50ng

100ng

250ng

500ng








bull Flexible





Proteomic Assays



Nucleic Acids








or IgM





Multiplex Detection

Detection


IgG

1 2 3 4





















Y Y

Y

IgG1 IgG2

IgG3

IgG4 IgA



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Detection



Antigen

1 2 3








Y Y Y

Y

Y

Y Y

Y Y

Y Y

Y



Y


Detection

Multiplex Detection


















compounds





compounds



SAPE SAPE














SAPE SAPE


Detection

Multiplex Detection












543-550






GTPases 827 271-282














Single Plex Assays

To Luminex

Multiplex

Assays






Detection

SAPE






HPV (high-risk

mucosal anogenital)




Quint et al 2009



HPV (high-risk

mucosal anogenital)



HPV (mucosal

anogenital)










Detection

RNA

Biotinylated cDNA








1 2





1

2

TAG-1

TAG-2



o Gene expression



o miRNA analysis


applications




ANNEAL

Ligate

PCR

TAG1

TAG2

cDNA or

gDNAPCR

HYBRIDIZE

amp

LABEL

dA

dG dT

dC -

ANNEAL

amp

Extend

TAG1

TAG2

HYBRIDIZE

amp

LABEL

gDNAPCR

SNP typing CF

CYP 450 RVP


CNV etc

RVP FAST GPP FAST

HYBRIDIZE

amp

LABEL

TAG1

TAG2 ANNEAL

amp

PCR

gDNA


30



2 hours



30 minutes



30 minutes





30 minutes







methods







TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

AAAAAAA RNA

AAAAAAA RNA

AAAAAAA RNA






TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

AAAAAAA RNA

AAAAAAA RNA

AAAAAAA RNA

cDNA

cDNA

cDNA




TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

cDNA

cDNA

cDNA




LMA Probe Design



Upstream LMA Probe

cDNA


Universal Primer 1

5rsquo 3rsquo












TAG

SAPE

Detection



lt3 Hours Setup











ANNEAL

Ligate

PCR

TAG1

TAG2

cDNA or

gDNAPCR

HYBRIDIZE

amp

LABEL





Upstream LMA Probes

T

Universal Primer 1

5rsquo

A

5rsquo

G



SNP

T



T

T


PCR Amplification

UP 1

P

No Amplification

B-UP 2



Universal Prime mix

UP 1

P

B-UP 2

UP 1

P

B-UP 2

Ligate

T T







TAG

SAPE

Detection



Bead Hyb 80 minutes







Incubation








Targeted PCR




Upstream LDR Probes

A T

G



SNP

A

A



A


Multiple

Ligation

cycles









TAG

SAPE

Detection






Forward primers

gDNA


5rsquo

3rsquo


A

T

5rsquo

3rsquo

G





Targeted PCR



A

5rsquo

3rsquo

G

5rsquo

3rsquo

T




A

5rsquo

3rsquo

G 5rsquo

3rsquo

T








xTAG


Detection

SAPE










xTAG primer

gDNA


Biotinylated primer

5rsquo 3rsquo

3rsquo

dATP dTTP dGTP dCTP

= Carbon Spacer











Detection

Sequence Detection

TAG


















Workflow

75



2 hours



30 minutes



30 minutes





30 minutes

76






0

500

1000

1500

2000

2500

3000

3500

4000


Sample

Sig

nal In

ten

sit

y (

MF

I)

let-7c

Let-7a

77


miR-107 vs miR-103

0

1000

2000

3000

4000

5000

6000


Sample

Sig

na

l In

ten

sit

y (

MF

I)

miR-103

miR-107









0

1000

2000

3000

4000

5000

6000

7000

8000

9000

10000

hsa-m

iR-1

07

hsa-m

iR-1

47

hsa-m

iR-1

81a

hsa-m

iR-1

92

hsa-m

iR-1

9b

hsa-m

iR-2

20a

hsa-m

iR-2

1hsa-m

iR-2

18

hsa-le

t-7c

hsa-m

iR-1

00

hsa-m

iR-1

25b

hsa-m

iR-

hsa-m

iR-2

05

hsa-m

iR-2

21

hsa-m

iR-2

00a

hsa-m

iR-2

00c

hsa-m

iR-2

06

hsa-m

iR-1

0a

hsa-m

iR-1

6

hsa-m

iR-9

hsa-m

iR-4

24

hsa-m

iR-2

0a

hsa-m

iR-2

0b

hsa-le

t-7a

hsa-m

iR-9

5hsa-m

iR-1

36

hsa-m

iR-1

95

hsa-m

iR-2

10

hsa-m

iR-2

12

hsa-m

iR-

hsa-m

iR-1

7

hsa-m

iR-1

22

hsa-m

iR-1

43

hsa-m

iR-1

45

hsa-m

iR-1

06a

hsa-m

iR-

hsa-m

iR-1

0b

hsa-m

iR-1

hsa-m

iR-2

23

hsa-m

iR-1

81b

hsa-m

iR-1

55

hsa-le

t-7g

hsa-le

t-7i

hsa-m

iR-9

6

hsa-m

iR-1

37

hsa-m

iR-1

85

hsa-m

iR-1

38

hsa-m

iR-1

98

hsa-m

iR-1

33a

hsa-m

iR-1

33b

hsa-m

iR-1

5a

hsa-m

iR-3

4a

hsa-m

iR-2

9a

hsa-m

iR-2

9b

hsa-m

iR-1

03

hsa-m

iR-2

22

hsa-m

iR-1

46a

cel-m

iR-3

9cel-m

iR-5

4

cel-m

iR-2

38

Regions

Ne

t S

ign

al In

ten

sit

y (

MF

I)

106a

17


Total RNA Samples

53-Plex Assay

miR-9 miR-122 miR-1

0

5000

10000

15000

20000

25000

Samples

Ne

t S

ign

al In

ten

sit

y (

MF

I)

miR-9

miR-122

miR-1






289704

12431797

4050

7044

12899

18030

24973

23884

21852

19249

11326

6573

3852

2471

0

5000

10000

15000

20000

25000

30000


Input Amount

Ne

t S

ign

al In

ten

sit

y (

MF

I)

200 amol


81


0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000



Ne

t S

ign

al in

ten

sit

y (

MF

I)

82

miR-133a

000

020

040

060

080

100

120


Total RNA Samples

Rela

tive C

han

ge t

o H

eart

FlexmiR v2

qRT-PCR

Tissue

Re

lati

ve

Ch

an

ge


LNP-miRNA

qRT-PCR

83

miRNA

Re

lati

ve

Ch

an

ge



0

5

10

15

20

25


miRNA

Rela

tive C

han

ge

FlexmiR v2

qRT-PCR

LNP-miRNA

qPCR

84



0

5000

10000

15000

20000

25000

30000

hsa-m

iR-1

07

hsa-m

iR-1

47

hsa-m

iR-1

81a

hsa-m

iR-1

92

hsa-m

iR-1

9b

hsa-m

iR-2

20a

hsa-m

iR-2

1hsa-m

iR-2

18

hsa-le

t-7c

hsa-m

iR-1

00

hsa-m

iR-1

25b

hsa-m

iR-

hsa-m

iR-2

05

hsa-m

iR-2

21

hsa-m

iR-2

00a

hsa-m

iR-2

00c

hsa-m

iR-2

06

hsa-m

iR-1

0a

hsa-m

iR-1

6hsa-m

iR-9

hsa-m

iR-4

24

hsa-m

iR-2

0a

hsa-m

iR-2

0b

hsa-le

t-7a

hsa-m

iR-9

5hsa-m

iR-1

36

hsa-m

iR-1

95

hsa-m

iR-2

10

hsa-m

iR-2

12

hsa-m

iR-

hsa-m

iR-1

7hsa-m

iR-1

22

hsa-m

iR-1

43

hsa-m

iR-1

45

hsa-m

ir-1

06a

hsa-m

iR-

hsa-m

iR-1

0b

hsa-m

iR-1

hsa-m

iR-2

23

hsa-m

iR-1

81b

hsa-le

t-155

hsa-le

t-7g

hsa-m

iR-7

ihsa-m

iR-9

6hsa-m

iR-1

37

hsa-m

iR-1

85

hsa-m

iR-1

38

hsa-m

iR-1

33a

hsa-m

iR-1

33b

hsa-m

iR-1

5a

hsa-m

iR-3

4a

hsa-m

iR-2

9a

hsa-m

iR-2

9b

hsa-m

iR-1

03

hsa-m

iR-2

22

hsa-m

iR-1

46a

hsa-m

iR-3

9cel-m

iR-3

9cel-m

iR-2

38

Regions

Ne

t S

ign

al in

ten

sit

y (

MF

I)

50ng

100ng

250ng

500ng








bull Flexible





Proteomic Assays



Nucleic Acids








or IgM





Multiplex Detection

Detection


IgG

1 2 3 4





















Y Y

Y

IgG1 IgG2

IgG3

IgG4 IgA



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Detection



Antigen

1 2 3








Y Y Y

Y

Y

Y Y

Y Y

Y Y

Y



Y


Detection

Multiplex Detection


















compounds





compounds



SAPE SAPE














SAPE SAPE


Detection

Multiplex Detection












543-550






GTPases 827 271-282














Single Plex Assays

To Luminex

Multiplex

Assays








HPV (high-risk

mucosal anogenital)




Quint et al 2009



HPV (high-risk

mucosal anogenital)



HPV (mucosal

anogenital)










Detection

RNA

Biotinylated cDNA








1 2





1

2

TAG-1

TAG-2



o Gene expression



o miRNA analysis


applications




ANNEAL

Ligate

PCR

TAG1

TAG2

cDNA or

gDNAPCR

HYBRIDIZE

amp

LABEL

dA

dG dT

dC -

ANNEAL

amp

Extend

TAG1

TAG2

HYBRIDIZE

amp

LABEL

gDNAPCR

SNP typing CF

CYP 450 RVP


CNV etc

RVP FAST GPP FAST

HYBRIDIZE

amp

LABEL

TAG1

TAG2 ANNEAL

amp

PCR

gDNA


30



2 hours



30 minutes



30 minutes





30 minutes







methods







TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

AAAAAAA RNA

AAAAAAA RNA

AAAAAAA RNA






TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

AAAAAAA RNA

AAAAAAA RNA

AAAAAAA RNA

cDNA

cDNA

cDNA




TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

cDNA

cDNA

cDNA




LMA Probe Design



Upstream LMA Probe

cDNA


Universal Primer 1

5rsquo 3rsquo












TAG

SAPE

Detection



lt3 Hours Setup











ANNEAL

Ligate

PCR

TAG1

TAG2

cDNA or

gDNAPCR

HYBRIDIZE

amp

LABEL





Upstream LMA Probes

T

Universal Primer 1

5rsquo

A

5rsquo

G



SNP

T



T

T


PCR Amplification

UP 1

P

No Amplification

B-UP 2



Universal Prime mix

UP 1

P

B-UP 2

UP 1

P

B-UP 2

Ligate

T T







TAG

SAPE

Detection



Bead Hyb 80 minutes







Incubation








Targeted PCR




Upstream LDR Probes

A T

G



SNP

A

A



A


Multiple

Ligation

cycles









TAG

SAPE

Detection






Forward primers

gDNA


5rsquo

3rsquo


A

T

5rsquo

3rsquo

G





Targeted PCR



A

5rsquo

3rsquo

G

5rsquo

3rsquo

T




A

5rsquo

3rsquo

G 5rsquo

3rsquo

T








xTAG


Detection

SAPE










xTAG primer

gDNA


Biotinylated primer

5rsquo 3rsquo

3rsquo

dATP dTTP dGTP dCTP

= Carbon Spacer











Detection

Sequence Detection

TAG


















Workflow

75



2 hours



30 minutes



30 minutes





30 minutes

76






0

500

1000

1500

2000

2500

3000

3500

4000


Sample

Sig

nal In

ten

sit

y (

MF

I)

let-7c

Let-7a

77


miR-107 vs miR-103

0

1000

2000

3000

4000

5000

6000


Sample

Sig

na

l In

ten

sit

y (

MF

I)

miR-103

miR-107









0

1000

2000

3000

4000

5000

6000

7000

8000

9000

10000

hsa-m

iR-1

07

hsa-m

iR-1

47

hsa-m

iR-1

81a

hsa-m

iR-1

92

hsa-m

iR-1

9b

hsa-m

iR-2

20a

hsa-m

iR-2

1hsa-m

iR-2

18

hsa-le

t-7c

hsa-m

iR-1

00

hsa-m

iR-1

25b

hsa-m

iR-

hsa-m

iR-2

05

hsa-m

iR-2

21

hsa-m

iR-2

00a

hsa-m

iR-2

00c

hsa-m

iR-2

06

hsa-m

iR-1

0a

hsa-m

iR-1

6

hsa-m

iR-9

hsa-m

iR-4

24

hsa-m

iR-2

0a

hsa-m

iR-2

0b

hsa-le

t-7a

hsa-m

iR-9

5hsa-m

iR-1

36

hsa-m

iR-1

95

hsa-m

iR-2

10

hsa-m

iR-2

12

hsa-m

iR-

hsa-m

iR-1

7

hsa-m

iR-1

22

hsa-m

iR-1

43

hsa-m

iR-1

45

hsa-m

iR-1

06a

hsa-m

iR-

hsa-m

iR-1

0b

hsa-m

iR-1

hsa-m

iR-2

23

hsa-m

iR-1

81b

hsa-m

iR-1

55

hsa-le

t-7g

hsa-le

t-7i

hsa-m

iR-9

6

hsa-m

iR-1

37

hsa-m

iR-1

85

hsa-m

iR-1

38

hsa-m

iR-1

98

hsa-m

iR-1

33a

hsa-m

iR-1

33b

hsa-m

iR-1

5a

hsa-m

iR-3

4a

hsa-m

iR-2

9a

hsa-m

iR-2

9b

hsa-m

iR-1

03

hsa-m

iR-2

22

hsa-m

iR-1

46a

cel-m

iR-3

9cel-m

iR-5

4

cel-m

iR-2

38

Regions

Ne

t S

ign

al In

ten

sit

y (

MF

I)

106a

17


Total RNA Samples

53-Plex Assay

miR-9 miR-122 miR-1

0

5000

10000

15000

20000

25000

Samples

Ne

t S

ign

al In

ten

sit

y (

MF

I)

miR-9

miR-122

miR-1






289704

12431797

4050

7044

12899

18030

24973

23884

21852

19249

11326

6573

3852

2471

0

5000

10000

15000

20000

25000

30000


Input Amount

Ne

t S

ign

al In

ten

sit

y (

MF

I)

200 amol


81


0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000



Ne

t S

ign

al in

ten

sit

y (

MF

I)

82

miR-133a

000

020

040

060

080

100

120


Total RNA Samples

Rela

tive C

han

ge t

o H

eart

FlexmiR v2

qRT-PCR

Tissue

Re

lati

ve

Ch

an

ge


LNP-miRNA

qRT-PCR

83

miRNA

Re

lati

ve

Ch

an

ge



0

5

10

15

20

25


miRNA

Rela

tive C

han

ge

FlexmiR v2

qRT-PCR

LNP-miRNA

qPCR

84



0

5000

10000

15000

20000

25000

30000

hsa-m

iR-1

07

hsa-m

iR-1

47

hsa-m

iR-1

81a

hsa-m

iR-1

92

hsa-m

iR-1

9b

hsa-m

iR-2

20a

hsa-m

iR-2

1hsa-m

iR-2

18

hsa-le

t-7c

hsa-m

iR-1

00

hsa-m

iR-1

25b

hsa-m

iR-

hsa-m

iR-2

05

hsa-m

iR-2

21

hsa-m

iR-2

00a

hsa-m

iR-2

00c

hsa-m

iR-2

06

hsa-m

iR-1

0a

hsa-m

iR-1

6hsa-m

iR-9

hsa-m

iR-4

24

hsa-m

iR-2

0a

hsa-m

iR-2

0b

hsa-le

t-7a

hsa-m

iR-9

5hsa-m

iR-1

36

hsa-m

iR-1

95

hsa-m

iR-2

10

hsa-m

iR-2

12

hsa-m

iR-

hsa-m

iR-1

7hsa-m

iR-1

22

hsa-m

iR-1

43

hsa-m

iR-1

45

hsa-m

ir-1

06a

hsa-m

iR-

hsa-m

iR-1

0b

hsa-m

iR-1

hsa-m

iR-2

23

hsa-m

iR-1

81b

hsa-le

t-155

hsa-le

t-7g

hsa-m

iR-7

ihsa-m

iR-9

6hsa-m

iR-1

37

hsa-m

iR-1

85

hsa-m

iR-1

38

hsa-m

iR-1

33a

hsa-m

iR-1

33b

hsa-m

iR-1

5a

hsa-m

iR-3

4a

hsa-m

iR-2

9a

hsa-m

iR-2

9b

hsa-m

iR-1

03

hsa-m

iR-2

22

hsa-m

iR-1

46a

hsa-m

iR-3

9cel-m

iR-3

9cel-m

iR-2

38

Regions

Ne

t S

ign

al in

ten

sit

y (

MF

I)

50ng

100ng

250ng

500ng








bull Flexible





Proteomic Assays



Nucleic Acids








or IgM





Multiplex Detection

Detection


IgG

1 2 3 4





















Y Y

Y

IgG1 IgG2

IgG3

IgG4 IgA



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Detection



Antigen

1 2 3








Y Y Y

Y

Y

Y Y

Y Y

Y Y

Y



Y


Detection

Multiplex Detection


















compounds





compounds



SAPE SAPE














SAPE SAPE


Detection

Multiplex Detection












543-550






GTPases 827 271-282














Single Plex Assays

To Luminex

Multiplex

Assays






HPV (high-risk

mucosal anogenital)




Quint et al 2009



HPV (high-risk

mucosal anogenital)



HPV (mucosal

anogenital)










Detection

RNA

Biotinylated cDNA








1 2





1

2

TAG-1

TAG-2



o Gene expression



o miRNA analysis


applications




ANNEAL

Ligate

PCR

TAG1

TAG2

cDNA or

gDNAPCR

HYBRIDIZE

amp

LABEL

dA

dG dT

dC -

ANNEAL

amp

Extend

TAG1

TAG2

HYBRIDIZE

amp

LABEL

gDNAPCR

SNP typing CF

CYP 450 RVP


CNV etc

RVP FAST GPP FAST

HYBRIDIZE

amp

LABEL

TAG1

TAG2 ANNEAL

amp

PCR

gDNA


30



2 hours



30 minutes



30 minutes





30 minutes







methods







TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

AAAAAAA RNA

AAAAAAA RNA

AAAAAAA RNA






TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

AAAAAAA RNA

AAAAAAA RNA

AAAAAAA RNA

cDNA

cDNA

cDNA




TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

cDNA

cDNA

cDNA




LMA Probe Design



Upstream LMA Probe

cDNA


Universal Primer 1

5rsquo 3rsquo












TAG

SAPE

Detection



lt3 Hours Setup











ANNEAL

Ligate

PCR

TAG1

TAG2

cDNA or

gDNAPCR

HYBRIDIZE

amp

LABEL





Upstream LMA Probes

T

Universal Primer 1

5rsquo

A

5rsquo

G



SNP

T



T

T


PCR Amplification

UP 1

P

No Amplification

B-UP 2



Universal Prime mix

UP 1

P

B-UP 2

UP 1

P

B-UP 2

Ligate

T T







TAG

SAPE

Detection



Bead Hyb 80 minutes







Incubation








Targeted PCR




Upstream LDR Probes

A T

G



SNP

A

A



A


Multiple

Ligation

cycles









TAG

SAPE

Detection






Forward primers

gDNA


5rsquo

3rsquo


A

T

5rsquo

3rsquo

G





Targeted PCR



A

5rsquo

3rsquo

G

5rsquo

3rsquo

T




A

5rsquo

3rsquo

G 5rsquo

3rsquo

T








xTAG


Detection

SAPE










xTAG primer

gDNA


Biotinylated primer

5rsquo 3rsquo

3rsquo

dATP dTTP dGTP dCTP

= Carbon Spacer











Detection

Sequence Detection

TAG


















Workflow

75



2 hours



30 minutes



30 minutes





30 minutes

76






0

500

1000

1500

2000

2500

3000

3500

4000


Sample

Sig

nal In

ten

sit

y (

MF

I)

let-7c

Let-7a

77


miR-107 vs miR-103

0

1000

2000

3000

4000

5000

6000


Sample

Sig

na

l In

ten

sit

y (

MF

I)

miR-103

miR-107









0

1000

2000

3000

4000

5000

6000

7000

8000

9000

10000

hsa-m

iR-1

07

hsa-m

iR-1

47

hsa-m

iR-1

81a

hsa-m

iR-1

92

hsa-m

iR-1

9b

hsa-m

iR-2

20a

hsa-m

iR-2

1hsa-m

iR-2

18

hsa-le

t-7c

hsa-m

iR-1

00

hsa-m

iR-1

25b

hsa-m

iR-

hsa-m

iR-2

05

hsa-m

iR-2

21

hsa-m

iR-2

00a

hsa-m

iR-2

00c

hsa-m

iR-2

06

hsa-m

iR-1

0a

hsa-m

iR-1

6

hsa-m

iR-9

hsa-m

iR-4

24

hsa-m

iR-2

0a

hsa-m

iR-2

0b

hsa-le

t-7a

hsa-m

iR-9

5hsa-m

iR-1

36

hsa-m

iR-1

95

hsa-m

iR-2

10

hsa-m

iR-2

12

hsa-m

iR-

hsa-m

iR-1

7

hsa-m

iR-1

22

hsa-m

iR-1

43

hsa-m

iR-1

45

hsa-m

iR-1

06a

hsa-m

iR-

hsa-m

iR-1

0b

hsa-m

iR-1

hsa-m

iR-2

23

hsa-m

iR-1

81b

hsa-m

iR-1

55

hsa-le

t-7g

hsa-le

t-7i

hsa-m

iR-9

6

hsa-m

iR-1

37

hsa-m

iR-1

85

hsa-m

iR-1

38

hsa-m

iR-1

98

hsa-m

iR-1

33a

hsa-m

iR-1

33b

hsa-m

iR-1

5a

hsa-m

iR-3

4a

hsa-m

iR-2

9a

hsa-m

iR-2

9b

hsa-m

iR-1

03

hsa-m

iR-2

22

hsa-m

iR-1

46a

cel-m

iR-3

9cel-m

iR-5

4

cel-m

iR-2

38

Regions

Ne

t S

ign

al In

ten

sit

y (

MF

I)

106a

17


Total RNA Samples

53-Plex Assay

miR-9 miR-122 miR-1

0

5000

10000

15000

20000

25000

Samples

Ne

t S

ign

al In

ten

sit

y (

MF

I)

miR-9

miR-122

miR-1






289704

12431797

4050

7044

12899

18030

24973

23884

21852

19249

11326

6573

3852

2471

0

5000

10000

15000

20000

25000

30000


Input Amount

Ne

t S

ign

al In

ten

sit

y (

MF

I)

200 amol


81


0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000



Ne

t S

ign

al in

ten

sit

y (

MF

I)

82

miR-133a

000

020

040

060

080

100

120


Total RNA Samples

Rela

tive C

han

ge t

o H

eart

FlexmiR v2

qRT-PCR

Tissue

Re

lati

ve

Ch

an

ge


LNP-miRNA

qRT-PCR

83

miRNA

Re

lati

ve

Ch

an

ge



0

5

10

15

20

25


miRNA

Rela

tive C

han

ge

FlexmiR v2

qRT-PCR

LNP-miRNA

qPCR

84



0

5000

10000

15000

20000

25000

30000

hsa-m

iR-1

07

hsa-m

iR-1

47

hsa-m

iR-1

81a

hsa-m

iR-1

92

hsa-m

iR-1

9b

hsa-m

iR-2

20a

hsa-m

iR-2

1hsa-m

iR-2

18

hsa-le

t-7c

hsa-m

iR-1

00

hsa-m

iR-1

25b

hsa-m

iR-

hsa-m

iR-2

05

hsa-m

iR-2

21

hsa-m

iR-2

00a

hsa-m

iR-2

00c

hsa-m

iR-2

06

hsa-m

iR-1

0a

hsa-m

iR-1

6hsa-m

iR-9

hsa-m

iR-4

24

hsa-m

iR-2

0a

hsa-m

iR-2

0b

hsa-le

t-7a

hsa-m

iR-9

5hsa-m

iR-1

36

hsa-m

iR-1

95

hsa-m

iR-2

10

hsa-m

iR-2

12

hsa-m

iR-

hsa-m

iR-1

7hsa-m

iR-1

22

hsa-m

iR-1

43

hsa-m

iR-1

45

hsa-m

ir-1

06a

hsa-m

iR-

hsa-m

iR-1

0b

hsa-m

iR-1

hsa-m

iR-2

23

hsa-m

iR-1

81b

hsa-le

t-155

hsa-le

t-7g

hsa-m

iR-7

ihsa-m

iR-9

6hsa-m

iR-1

37

hsa-m

iR-1

85

hsa-m

iR-1

38

hsa-m

iR-1

33a

hsa-m

iR-1

33b

hsa-m

iR-1

5a

hsa-m

iR-3

4a

hsa-m

iR-2

9a

hsa-m

iR-2

9b

hsa-m

iR-1

03

hsa-m

iR-2

22

hsa-m

iR-1

46a

hsa-m

iR-3

9cel-m

iR-3

9cel-m

iR-2

38

Regions

Ne

t S

ign

al in

ten

sit

y (

MF

I)

50ng

100ng

250ng

500ng








bull Flexible





Proteomic Assays



Nucleic Acids








or IgM





Multiplex Detection

Detection


IgG

1 2 3 4





















Y Y

Y

IgG1 IgG2

IgG3

IgG4 IgA



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Detection



Antigen

1 2 3








Y Y Y

Y

Y

Y Y

Y Y

Y Y

Y



Y


Detection

Multiplex Detection


















compounds





compounds



SAPE SAPE














SAPE SAPE


Detection

Multiplex Detection












543-550






GTPases 827 271-282














Single Plex Assays

To Luminex

Multiplex

Assays








Detection

RNA

Biotinylated cDNA








1 2





1

2

TAG-1

TAG-2



o Gene expression



o miRNA analysis


applications




ANNEAL

Ligate

PCR

TAG1

TAG2

cDNA or

gDNAPCR

HYBRIDIZE

amp

LABEL

dA

dG dT

dC -

ANNEAL

amp

Extend

TAG1

TAG2

HYBRIDIZE

amp

LABEL

gDNAPCR

SNP typing CF

CYP 450 RVP


CNV etc

RVP FAST GPP FAST

HYBRIDIZE

amp

LABEL

TAG1

TAG2 ANNEAL

amp

PCR

gDNA


30



2 hours



30 minutes



30 minutes





30 minutes







methods







TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

AAAAAAA RNA

AAAAAAA RNA

AAAAAAA RNA






TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

AAAAAAA RNA

AAAAAAA RNA

AAAAAAA RNA

cDNA

cDNA

cDNA




TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

cDNA

cDNA

cDNA




LMA Probe Design



Upstream LMA Probe

cDNA


Universal Primer 1

5rsquo 3rsquo












TAG

SAPE

Detection



lt3 Hours Setup











ANNEAL

Ligate

PCR

TAG1

TAG2

cDNA or

gDNAPCR

HYBRIDIZE

amp

LABEL





Upstream LMA Probes

T

Universal Primer 1

5rsquo

A

5rsquo

G



SNP

T



T

T


PCR Amplification

UP 1

P

No Amplification

B-UP 2



Universal Prime mix

UP 1

P

B-UP 2

UP 1

P

B-UP 2

Ligate

T T







TAG

SAPE

Detection



Bead Hyb 80 minutes







Incubation








Targeted PCR




Upstream LDR Probes

A T

G



SNP

A

A



A


Multiple

Ligation

cycles









TAG

SAPE

Detection






Forward primers

gDNA


5rsquo

3rsquo


A

T

5rsquo

3rsquo

G





Targeted PCR



A

5rsquo

3rsquo

G

5rsquo

3rsquo

T




A

5rsquo

3rsquo

G 5rsquo

3rsquo

T








xTAG


Detection

SAPE










xTAG primer

gDNA


Biotinylated primer

5rsquo 3rsquo

3rsquo

dATP dTTP dGTP dCTP

= Carbon Spacer











Detection

Sequence Detection

TAG


















Workflow

75



2 hours



30 minutes



30 minutes





30 minutes

76






0

500

1000

1500

2000

2500

3000

3500

4000


Sample

Sig

nal In

ten

sit

y (

MF

I)

let-7c

Let-7a

77


miR-107 vs miR-103

0

1000

2000

3000

4000

5000

6000


Sample

Sig

na

l In

ten

sit

y (

MF

I)

miR-103

miR-107









0

1000

2000

3000

4000

5000

6000

7000

8000

9000

10000

hsa-m

iR-1

07

hsa-m

iR-1

47

hsa-m

iR-1

81a

hsa-m

iR-1

92

hsa-m

iR-1

9b

hsa-m

iR-2

20a

hsa-m

iR-2

1hsa-m

iR-2

18

hsa-le

t-7c

hsa-m

iR-1

00

hsa-m

iR-1

25b

hsa-m

iR-

hsa-m

iR-2

05

hsa-m

iR-2

21

hsa-m

iR-2

00a

hsa-m

iR-2

00c

hsa-m

iR-2

06

hsa-m

iR-1

0a

hsa-m

iR-1

6

hsa-m

iR-9

hsa-m

iR-4

24

hsa-m

iR-2

0a

hsa-m

iR-2

0b

hsa-le

t-7a

hsa-m

iR-9

5hsa-m

iR-1

36

hsa-m

iR-1

95

hsa-m

iR-2

10

hsa-m

iR-2

12

hsa-m

iR-

hsa-m

iR-1

7

hsa-m

iR-1

22

hsa-m

iR-1

43

hsa-m

iR-1

45

hsa-m

iR-1

06a

hsa-m

iR-

hsa-m

iR-1

0b

hsa-m

iR-1

hsa-m

iR-2

23

hsa-m

iR-1

81b

hsa-m

iR-1

55

hsa-le

t-7g

hsa-le

t-7i

hsa-m

iR-9

6

hsa-m

iR-1

37

hsa-m

iR-1

85

hsa-m

iR-1

38

hsa-m

iR-1

98

hsa-m

iR-1

33a

hsa-m

iR-1

33b

hsa-m

iR-1

5a

hsa-m

iR-3

4a

hsa-m

iR-2

9a

hsa-m

iR-2

9b

hsa-m

iR-1

03

hsa-m

iR-2

22

hsa-m

iR-1

46a

cel-m

iR-3

9cel-m

iR-5

4

cel-m

iR-2

38

Regions

Ne

t S

ign

al In

ten

sit

y (

MF

I)

106a

17


Total RNA Samples

53-Plex Assay

miR-9 miR-122 miR-1

0

5000

10000

15000

20000

25000

Samples

Ne

t S

ign

al In

ten

sit

y (

MF

I)

miR-9

miR-122

miR-1






289704

12431797

4050

7044

12899

18030

24973

23884

21852

19249

11326

6573

3852

2471

0

5000

10000

15000

20000

25000

30000


Input Amount

Ne

t S

ign

al In

ten

sit

y (

MF

I)

200 amol


81


0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000



Ne

t S

ign

al in

ten

sit

y (

MF

I)

82

miR-133a

000

020

040

060

080

100

120


Total RNA Samples

Rela

tive C

han

ge t

o H

eart

FlexmiR v2

qRT-PCR

Tissue

Re

lati

ve

Ch

an

ge


LNP-miRNA

qRT-PCR

83

miRNA

Re

lati

ve

Ch

an

ge



0

5

10

15

20

25


miRNA

Rela

tive C

han

ge

FlexmiR v2

qRT-PCR

LNP-miRNA

qPCR

84



0

5000

10000

15000

20000

25000

30000

hsa-m

iR-1

07

hsa-m

iR-1

47

hsa-m

iR-1

81a

hsa-m

iR-1

92

hsa-m

iR-1

9b

hsa-m

iR-2

20a

hsa-m

iR-2

1hsa-m

iR-2

18

hsa-le

t-7c

hsa-m

iR-1

00

hsa-m

iR-1

25b

hsa-m

iR-

hsa-m

iR-2

05

hsa-m

iR-2

21

hsa-m

iR-2

00a

hsa-m

iR-2

00c

hsa-m

iR-2

06

hsa-m

iR-1

0a

hsa-m

iR-1

6hsa-m

iR-9

hsa-m

iR-4

24

hsa-m

iR-2

0a

hsa-m

iR-2

0b

hsa-le

t-7a

hsa-m

iR-9

5hsa-m

iR-1

36

hsa-m

iR-1

95

hsa-m

iR-2

10

hsa-m

iR-2

12

hsa-m

iR-

hsa-m

iR-1

7hsa-m

iR-1

22

hsa-m

iR-1

43

hsa-m

iR-1

45

hsa-m

ir-1

06a

hsa-m

iR-

hsa-m

iR-1

0b

hsa-m

iR-1

hsa-m

iR-2

23

hsa-m

iR-1

81b

hsa-le

t-155

hsa-le

t-7g

hsa-m

iR-7

ihsa-m

iR-9

6hsa-m

iR-1

37

hsa-m

iR-1

85

hsa-m

iR-1

38

hsa-m

iR-1

33a

hsa-m

iR-1

33b

hsa-m

iR-1

5a

hsa-m

iR-3

4a

hsa-m

iR-2

9a

hsa-m

iR-2

9b

hsa-m

iR-1

03

hsa-m

iR-2

22

hsa-m

iR-1

46a

hsa-m

iR-3

9cel-m

iR-3

9cel-m

iR-2

38

Regions

Ne

t S

ign

al in

ten

sit

y (

MF

I)

50ng

100ng

250ng

500ng








bull Flexible





Proteomic Assays



Nucleic Acids








or IgM





Multiplex Detection

Detection


IgG

1 2 3 4





















Y Y

Y

IgG1 IgG2

IgG3

IgG4 IgA



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Detection



Antigen

1 2 3








Y Y Y

Y

Y

Y Y

Y Y

Y Y

Y



Y


Detection

Multiplex Detection


















compounds





compounds



SAPE SAPE














SAPE SAPE


Detection

Multiplex Detection












543-550






GTPases 827 271-282














Single Plex Assays

To Luminex

Multiplex

Assays






Detection

RNA

Biotinylated cDNA








1 2





1

2

TAG-1

TAG-2



o Gene expression



o miRNA analysis


applications




ANNEAL

Ligate

PCR

TAG1

TAG2

cDNA or

gDNAPCR

HYBRIDIZE

amp

LABEL

dA

dG dT

dC -

ANNEAL

amp

Extend

TAG1

TAG2

HYBRIDIZE

amp

LABEL

gDNAPCR

SNP typing CF

CYP 450 RVP


CNV etc

RVP FAST GPP FAST

HYBRIDIZE

amp

LABEL

TAG1

TAG2 ANNEAL

amp

PCR

gDNA


30



2 hours



30 minutes



30 minutes





30 minutes







methods







TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

AAAAAAA RNA

AAAAAAA RNA

AAAAAAA RNA






TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

AAAAAAA RNA

AAAAAAA RNA

AAAAAAA RNA

cDNA

cDNA

cDNA




TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

cDNA

cDNA

cDNA




LMA Probe Design



Upstream LMA Probe

cDNA


Universal Primer 1

5rsquo 3rsquo












TAG

SAPE

Detection



lt3 Hours Setup











ANNEAL

Ligate

PCR

TAG1

TAG2

cDNA or

gDNAPCR

HYBRIDIZE

amp

LABEL





Upstream LMA Probes

T

Universal Primer 1

5rsquo

A

5rsquo

G



SNP

T



T

T


PCR Amplification

UP 1

P

No Amplification

B-UP 2



Universal Prime mix

UP 1

P

B-UP 2

UP 1

P

B-UP 2

Ligate

T T







TAG

SAPE

Detection



Bead Hyb 80 minutes







Incubation








Targeted PCR




Upstream LDR Probes

A T

G



SNP

A

A



A


Multiple

Ligation

cycles









TAG

SAPE

Detection






Forward primers

gDNA


5rsquo

3rsquo


A

T

5rsquo

3rsquo

G





Targeted PCR



A

5rsquo

3rsquo

G

5rsquo

3rsquo

T




A

5rsquo

3rsquo

G 5rsquo

3rsquo

T








xTAG


Detection

SAPE










xTAG primer

gDNA


Biotinylated primer

5rsquo 3rsquo

3rsquo

dATP dTTP dGTP dCTP

= Carbon Spacer











Detection

Sequence Detection

TAG


















Workflow

75



2 hours



30 minutes



30 minutes





30 minutes

76






0

500

1000

1500

2000

2500

3000

3500

4000


Sample

Sig

nal In

ten

sit

y (

MF

I)

let-7c

Let-7a

77


miR-107 vs miR-103

0

1000

2000

3000

4000

5000

6000


Sample

Sig

na

l In

ten

sit

y (

MF

I)

miR-103

miR-107









0

1000

2000

3000

4000

5000

6000

7000

8000

9000

10000

hsa-m

iR-1

07

hsa-m

iR-1

47

hsa-m

iR-1

81a

hsa-m

iR-1

92

hsa-m

iR-1

9b

hsa-m

iR-2

20a

hsa-m

iR-2

1hsa-m

iR-2

18

hsa-le

t-7c

hsa-m

iR-1

00

hsa-m

iR-1

25b

hsa-m

iR-

hsa-m

iR-2

05

hsa-m

iR-2

21

hsa-m

iR-2

00a

hsa-m

iR-2

00c

hsa-m

iR-2

06

hsa-m

iR-1

0a

hsa-m

iR-1

6

hsa-m

iR-9

hsa-m

iR-4

24

hsa-m

iR-2

0a

hsa-m

iR-2

0b

hsa-le

t-7a

hsa-m

iR-9

5hsa-m

iR-1

36

hsa-m

iR-1

95

hsa-m

iR-2

10

hsa-m

iR-2

12

hsa-m

iR-

hsa-m

iR-1

7

hsa-m

iR-1

22

hsa-m

iR-1

43

hsa-m

iR-1

45

hsa-m

iR-1

06a

hsa-m

iR-

hsa-m

iR-1

0b

hsa-m

iR-1

hsa-m

iR-2

23

hsa-m

iR-1

81b

hsa-m

iR-1

55

hsa-le

t-7g

hsa-le

t-7i

hsa-m

iR-9

6

hsa-m

iR-1

37

hsa-m

iR-1

85

hsa-m

iR-1

38

hsa-m

iR-1

98

hsa-m

iR-1

33a

hsa-m

iR-1

33b

hsa-m

iR-1

5a

hsa-m

iR-3

4a

hsa-m

iR-2

9a

hsa-m

iR-2

9b

hsa-m

iR-1

03

hsa-m

iR-2

22

hsa-m

iR-1

46a

cel-m

iR-3

9cel-m

iR-5

4

cel-m

iR-2

38

Regions

Ne

t S

ign

al In

ten

sit

y (

MF

I)

106a

17


Total RNA Samples

53-Plex Assay

miR-9 miR-122 miR-1

0

5000

10000

15000

20000

25000

Samples

Ne

t S

ign

al In

ten

sit

y (

MF

I)

miR-9

miR-122

miR-1






289704

12431797

4050

7044

12899

18030

24973

23884

21852

19249

11326

6573

3852

2471

0

5000

10000

15000

20000

25000

30000


Input Amount

Ne

t S

ign

al In

ten

sit

y (

MF

I)

200 amol


81


0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000



Ne

t S

ign

al in

ten

sit

y (

MF

I)

82

miR-133a

000

020

040

060

080

100

120


Total RNA Samples

Rela

tive C

han

ge t

o H

eart

FlexmiR v2

qRT-PCR

Tissue

Re

lati

ve

Ch

an

ge


LNP-miRNA

qRT-PCR

83

miRNA

Re

lati

ve

Ch

an

ge



0

5

10

15

20

25


miRNA

Rela

tive C

han

ge

FlexmiR v2

qRT-PCR

LNP-miRNA

qPCR

84



0

5000

10000

15000

20000

25000

30000

hsa-m

iR-1

07

hsa-m

iR-1

47

hsa-m

iR-1

81a

hsa-m

iR-1

92

hsa-m

iR-1

9b

hsa-m

iR-2

20a

hsa-m

iR-2

1hsa-m

iR-2

18

hsa-le

t-7c

hsa-m

iR-1

00

hsa-m

iR-1

25b

hsa-m

iR-

hsa-m

iR-2

05

hsa-m

iR-2

21

hsa-m

iR-2

00a

hsa-m

iR-2

00c

hsa-m

iR-2

06

hsa-m

iR-1

0a

hsa-m

iR-1

6hsa-m

iR-9

hsa-m

iR-4

24

hsa-m

iR-2

0a

hsa-m

iR-2

0b

hsa-le

t-7a

hsa-m

iR-9

5hsa-m

iR-1

36

hsa-m

iR-1

95

hsa-m

iR-2

10

hsa-m

iR-2

12

hsa-m

iR-

hsa-m

iR-1

7hsa-m

iR-1

22

hsa-m

iR-1

43

hsa-m

iR-1

45

hsa-m

ir-1

06a

hsa-m

iR-

hsa-m

iR-1

0b

hsa-m

iR-1

hsa-m

iR-2

23

hsa-m

iR-1

81b

hsa-le

t-155

hsa-le

t-7g

hsa-m

iR-7

ihsa-m

iR-9

6hsa-m

iR-1

37

hsa-m

iR-1

85

hsa-m

iR-1

38

hsa-m

iR-1

33a

hsa-m

iR-1

33b

hsa-m

iR-1

5a

hsa-m

iR-3

4a

hsa-m

iR-2

9a

hsa-m

iR-2

9b

hsa-m

iR-1

03

hsa-m

iR-2

22

hsa-m

iR-1

46a

hsa-m

iR-3

9cel-m

iR-3

9cel-m

iR-2

38

Regions

Ne

t S

ign

al in

ten

sit

y (

MF

I)

50ng

100ng

250ng

500ng








bull Flexible





Proteomic Assays



Nucleic Acids








or IgM





Multiplex Detection

Detection


IgG

1 2 3 4





















Y Y

Y

IgG1 IgG2

IgG3

IgG4 IgA



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Detection



Antigen

1 2 3








Y Y Y

Y

Y

Y Y

Y Y

Y Y

Y



Y


Detection

Multiplex Detection


















compounds





compounds



SAPE SAPE














SAPE SAPE


Detection

Multiplex Detection












543-550






GTPases 827 271-282














Single Plex Assays

To Luminex

Multiplex

Assays










1 2





1

2

TAG-1

TAG-2



o Gene expression



o miRNA analysis


applications




ANNEAL

Ligate

PCR

TAG1

TAG2

cDNA or

gDNAPCR

HYBRIDIZE

amp

LABEL

dA

dG dT

dC -

ANNEAL

amp

Extend

TAG1

TAG2

HYBRIDIZE

amp

LABEL

gDNAPCR

SNP typing CF

CYP 450 RVP


CNV etc

RVP FAST GPP FAST

HYBRIDIZE

amp

LABEL

TAG1

TAG2 ANNEAL

amp

PCR

gDNA


30



2 hours



30 minutes



30 minutes





30 minutes







methods







TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

AAAAAAA RNA

AAAAAAA RNA

AAAAAAA RNA






TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

AAAAAAA RNA

AAAAAAA RNA

AAAAAAA RNA

cDNA

cDNA

cDNA




TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

cDNA

cDNA

cDNA




LMA Probe Design



Upstream LMA Probe

cDNA


Universal Primer 1

5rsquo 3rsquo












TAG

SAPE

Detection



lt3 Hours Setup











ANNEAL

Ligate

PCR

TAG1

TAG2

cDNA or

gDNAPCR

HYBRIDIZE

amp

LABEL





Upstream LMA Probes

T

Universal Primer 1

5rsquo

A

5rsquo

G



SNP

T



T

T


PCR Amplification

UP 1

P

No Amplification

B-UP 2



Universal Prime mix

UP 1

P

B-UP 2

UP 1

P

B-UP 2

Ligate

T T







TAG

SAPE

Detection



Bead Hyb 80 minutes







Incubation








Targeted PCR




Upstream LDR Probes

A T

G



SNP

A

A



A


Multiple

Ligation

cycles









TAG

SAPE

Detection






Forward primers

gDNA


5rsquo

3rsquo


A

T

5rsquo

3rsquo

G





Targeted PCR



A

5rsquo

3rsquo

G

5rsquo

3rsquo

T




A

5rsquo

3rsquo

G 5rsquo

3rsquo

T








xTAG


Detection

SAPE










xTAG primer

gDNA


Biotinylated primer

5rsquo 3rsquo

3rsquo

dATP dTTP dGTP dCTP

= Carbon Spacer











Detection

Sequence Detection

TAG


















Workflow

75



2 hours



30 minutes



30 minutes





30 minutes

76






0

500

1000

1500

2000

2500

3000

3500

4000


Sample

Sig

nal In

ten

sit

y (

MF

I)

let-7c

Let-7a

77


miR-107 vs miR-103

0

1000

2000

3000

4000

5000

6000


Sample

Sig

na

l In

ten

sit

y (

MF

I)

miR-103

miR-107









0

1000

2000

3000

4000

5000

6000

7000

8000

9000

10000

hsa-m

iR-1

07

hsa-m

iR-1

47

hsa-m

iR-1

81a

hsa-m

iR-1

92

hsa-m

iR-1

9b

hsa-m

iR-2

20a

hsa-m

iR-2

1hsa-m

iR-2

18

hsa-le

t-7c

hsa-m

iR-1

00

hsa-m

iR-1

25b

hsa-m

iR-

hsa-m

iR-2

05

hsa-m

iR-2

21

hsa-m

iR-2

00a

hsa-m

iR-2

00c

hsa-m

iR-2

06

hsa-m

iR-1

0a

hsa-m

iR-1

6

hsa-m

iR-9

hsa-m

iR-4

24

hsa-m

iR-2

0a

hsa-m

iR-2

0b

hsa-le

t-7a

hsa-m

iR-9

5hsa-m

iR-1

36

hsa-m

iR-1

95

hsa-m

iR-2

10

hsa-m

iR-2

12

hsa-m

iR-

hsa-m

iR-1

7

hsa-m

iR-1

22

hsa-m

iR-1

43

hsa-m

iR-1

45

hsa-m

iR-1

06a

hsa-m

iR-

hsa-m

iR-1

0b

hsa-m

iR-1

hsa-m

iR-2

23

hsa-m

iR-1

81b

hsa-m

iR-1

55

hsa-le

t-7g

hsa-le

t-7i

hsa-m

iR-9

6

hsa-m

iR-1

37

hsa-m

iR-1

85

hsa-m

iR-1

38

hsa-m

iR-1

98

hsa-m

iR-1

33a

hsa-m

iR-1

33b

hsa-m

iR-1

5a

hsa-m

iR-3

4a

hsa-m

iR-2

9a

hsa-m

iR-2

9b

hsa-m

iR-1

03

hsa-m

iR-2

22

hsa-m

iR-1

46a

cel-m

iR-3

9cel-m

iR-5

4

cel-m

iR-2

38

Regions

Ne

t S

ign

al In

ten

sit

y (

MF

I)

106a

17


Total RNA Samples

53-Plex Assay

miR-9 miR-122 miR-1

0

5000

10000

15000

20000

25000

Samples

Ne

t S

ign

al In

ten

sit

y (

MF

I)

miR-9

miR-122

miR-1






289704

12431797

4050

7044

12899

18030

24973

23884

21852

19249

11326

6573

3852

2471

0

5000

10000

15000

20000

25000

30000


Input Amount

Ne

t S

ign

al In

ten

sit

y (

MF

I)

200 amol


81


0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000



Ne

t S

ign

al in

ten

sit

y (

MF

I)

82

miR-133a

000

020

040

060

080

100

120


Total RNA Samples

Rela

tive C

han

ge t

o H

eart

FlexmiR v2

qRT-PCR

Tissue

Re

lati

ve

Ch

an

ge


LNP-miRNA

qRT-PCR

83

miRNA

Re

lati

ve

Ch

an

ge



0

5

10

15

20

25


miRNA

Rela

tive C

han

ge

FlexmiR v2

qRT-PCR

LNP-miRNA

qPCR

84



0

5000

10000

15000

20000

25000

30000

hsa-m

iR-1

07

hsa-m

iR-1

47

hsa-m

iR-1

81a

hsa-m

iR-1

92

hsa-m

iR-1

9b

hsa-m

iR-2

20a

hsa-m

iR-2

1hsa-m

iR-2

18

hsa-le

t-7c

hsa-m

iR-1

00

hsa-m

iR-1

25b

hsa-m

iR-

hsa-m

iR-2

05

hsa-m

iR-2

21

hsa-m

iR-2

00a

hsa-m

iR-2

00c

hsa-m

iR-2

06

hsa-m

iR-1

0a

hsa-m

iR-1

6hsa-m

iR-9

hsa-m

iR-4

24

hsa-m

iR-2

0a

hsa-m

iR-2

0b

hsa-le

t-7a

hsa-m

iR-9

5hsa-m

iR-1

36

hsa-m

iR-1

95

hsa-m

iR-2

10

hsa-m

iR-2

12

hsa-m

iR-

hsa-m

iR-1

7hsa-m

iR-1

22

hsa-m

iR-1

43

hsa-m

iR-1

45

hsa-m

ir-1

06a

hsa-m

iR-

hsa-m

iR-1

0b

hsa-m

iR-1

hsa-m

iR-2

23

hsa-m

iR-1

81b

hsa-le

t-155

hsa-le

t-7g

hsa-m

iR-7

ihsa-m

iR-9

6hsa-m

iR-1

37

hsa-m

iR-1

85

hsa-m

iR-1

38

hsa-m

iR-1

33a

hsa-m

iR-1

33b

hsa-m

iR-1

5a

hsa-m

iR-3

4a

hsa-m

iR-2

9a

hsa-m

iR-2

9b

hsa-m

iR-1

03

hsa-m

iR-2

22

hsa-m

iR-1

46a

hsa-m

iR-3

9cel-m

iR-3

9cel-m

iR-2

38

Regions

Ne

t S

ign

al in

ten

sit

y (

MF

I)

50ng

100ng

250ng

500ng








bull Flexible





Proteomic Assays



Nucleic Acids








or IgM





Multiplex Detection

Detection


IgG

1 2 3 4





















Y Y

Y

IgG1 IgG2

IgG3

IgG4 IgA



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Detection



Antigen

1 2 3








Y Y Y

Y

Y

Y Y

Y Y

Y Y

Y



Y


Detection

Multiplex Detection


















compounds





compounds



SAPE SAPE














SAPE SAPE


Detection

Multiplex Detection












543-550






GTPases 827 271-282














Single Plex Assays

To Luminex

Multiplex

Assays








1 2





1

2

TAG-1

TAG-2



o Gene expression



o miRNA analysis


applications




ANNEAL

Ligate

PCR

TAG1

TAG2

cDNA or

gDNAPCR

HYBRIDIZE

amp

LABEL

dA

dG dT

dC -

ANNEAL

amp

Extend

TAG1

TAG2

HYBRIDIZE

amp

LABEL

gDNAPCR

SNP typing CF

CYP 450 RVP


CNV etc

RVP FAST GPP FAST

HYBRIDIZE

amp

LABEL

TAG1

TAG2 ANNEAL

amp

PCR

gDNA


30



2 hours



30 minutes



30 minutes





30 minutes







methods







TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

AAAAAAA RNA

AAAAAAA RNA

AAAAAAA RNA






TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

AAAAAAA RNA

AAAAAAA RNA

AAAAAAA RNA

cDNA

cDNA

cDNA




TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

cDNA

cDNA

cDNA




LMA Probe Design



Upstream LMA Probe

cDNA


Universal Primer 1

5rsquo 3rsquo












TAG

SAPE

Detection



lt3 Hours Setup











ANNEAL

Ligate

PCR

TAG1

TAG2

cDNA or

gDNAPCR

HYBRIDIZE

amp

LABEL





Upstream LMA Probes

T

Universal Primer 1

5rsquo

A

5rsquo

G



SNP

T



T

T


PCR Amplification

UP 1

P

No Amplification

B-UP 2



Universal Prime mix

UP 1

P

B-UP 2

UP 1

P

B-UP 2

Ligate

T T







TAG

SAPE

Detection



Bead Hyb 80 minutes







Incubation








Targeted PCR




Upstream LDR Probes

A T

G



SNP

A

A



A


Multiple

Ligation

cycles









TAG

SAPE

Detection






Forward primers

gDNA


5rsquo

3rsquo


A

T

5rsquo

3rsquo

G





Targeted PCR



A

5rsquo

3rsquo

G

5rsquo

3rsquo

T




A

5rsquo

3rsquo

G 5rsquo

3rsquo

T








xTAG


Detection

SAPE










xTAG primer

gDNA


Biotinylated primer

5rsquo 3rsquo

3rsquo

dATP dTTP dGTP dCTP

= Carbon Spacer











Detection

Sequence Detection

TAG


















Workflow

75



2 hours



30 minutes



30 minutes





30 minutes

76






0

500

1000

1500

2000

2500

3000

3500

4000


Sample

Sig

nal In

ten

sit

y (

MF

I)

let-7c

Let-7a

77


miR-107 vs miR-103

0

1000

2000

3000

4000

5000

6000


Sample

Sig

na

l In

ten

sit

y (

MF

I)

miR-103

miR-107









0

1000

2000

3000

4000

5000

6000

7000

8000

9000

10000

hsa-m

iR-1

07

hsa-m

iR-1

47

hsa-m

iR-1

81a

hsa-m

iR-1

92

hsa-m

iR-1

9b

hsa-m

iR-2

20a

hsa-m

iR-2

1hsa-m

iR-2

18

hsa-le

t-7c

hsa-m

iR-1

00

hsa-m

iR-1

25b

hsa-m

iR-

hsa-m

iR-2

05

hsa-m

iR-2

21

hsa-m

iR-2

00a

hsa-m

iR-2

00c

hsa-m

iR-2

06

hsa-m

iR-1

0a

hsa-m

iR-1

6

hsa-m

iR-9

hsa-m

iR-4

24

hsa-m

iR-2

0a

hsa-m

iR-2

0b

hsa-le

t-7a

hsa-m

iR-9

5hsa-m

iR-1

36

hsa-m

iR-1

95

hsa-m

iR-2

10

hsa-m

iR-2

12

hsa-m

iR-

hsa-m

iR-1

7

hsa-m

iR-1

22

hsa-m

iR-1

43

hsa-m

iR-1

45

hsa-m

iR-1

06a

hsa-m

iR-

hsa-m

iR-1

0b

hsa-m

iR-1

hsa-m

iR-2

23

hsa-m

iR-1

81b

hsa-m

iR-1

55

hsa-le

t-7g

hsa-le

t-7i

hsa-m

iR-9

6

hsa-m

iR-1

37

hsa-m

iR-1

85

hsa-m

iR-1

38

hsa-m

iR-1

98

hsa-m

iR-1

33a

hsa-m

iR-1

33b

hsa-m

iR-1

5a

hsa-m

iR-3

4a

hsa-m

iR-2

9a

hsa-m

iR-2

9b

hsa-m

iR-1

03

hsa-m

iR-2

22

hsa-m

iR-1

46a

cel-m

iR-3

9cel-m

iR-5

4

cel-m

iR-2

38

Regions

Ne

t S

ign

al In

ten

sit

y (

MF

I)

106a

17


Total RNA Samples

53-Plex Assay

miR-9 miR-122 miR-1

0

5000

10000

15000

20000

25000

Samples

Ne

t S

ign

al In

ten

sit

y (

MF

I)

miR-9

miR-122

miR-1






289704

12431797

4050

7044

12899

18030

24973

23884

21852

19249

11326

6573

3852

2471

0

5000

10000

15000

20000

25000

30000


Input Amount

Ne

t S

ign

al In

ten

sit

y (

MF

I)

200 amol


81


0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000



Ne

t S

ign

al in

ten

sit

y (

MF

I)

82

miR-133a

000

020

040

060

080

100

120


Total RNA Samples

Rela

tive C

han

ge t

o H

eart

FlexmiR v2

qRT-PCR

Tissue

Re

lati

ve

Ch

an

ge


LNP-miRNA

qRT-PCR

83

miRNA

Re

lati

ve

Ch

an

ge



0

5

10

15

20

25


miRNA

Rela

tive C

han

ge

FlexmiR v2

qRT-PCR

LNP-miRNA

qPCR

84



0

5000

10000

15000

20000

25000

30000

hsa-m

iR-1

07

hsa-m

iR-1

47

hsa-m

iR-1

81a

hsa-m

iR-1

92

hsa-m

iR-1

9b

hsa-m

iR-2

20a

hsa-m

iR-2

1hsa-m

iR-2

18

hsa-le

t-7c

hsa-m

iR-1

00

hsa-m

iR-1

25b

hsa-m

iR-

hsa-m

iR-2

05

hsa-m

iR-2

21

hsa-m

iR-2

00a

hsa-m

iR-2

00c

hsa-m

iR-2

06

hsa-m

iR-1

0a

hsa-m

iR-1

6hsa-m

iR-9

hsa-m

iR-4

24

hsa-m

iR-2

0a

hsa-m

iR-2

0b

hsa-le

t-7a

hsa-m

iR-9

5hsa-m

iR-1

36

hsa-m

iR-1

95

hsa-m

iR-2

10

hsa-m

iR-2

12

hsa-m

iR-

hsa-m

iR-1

7hsa-m

iR-1

22

hsa-m

iR-1

43

hsa-m

iR-1

45

hsa-m

ir-1

06a

hsa-m

iR-

hsa-m

iR-1

0b

hsa-m

iR-1

hsa-m

iR-2

23

hsa-m

iR-1

81b

hsa-le

t-155

hsa-le

t-7g

hsa-m

iR-7

ihsa-m

iR-9

6hsa-m

iR-1

37

hsa-m

iR-1

85

hsa-m

iR-1

38

hsa-m

iR-1

33a

hsa-m

iR-1

33b

hsa-m

iR-1

5a

hsa-m

iR-3

4a

hsa-m

iR-2

9a

hsa-m

iR-2

9b

hsa-m

iR-1

03

hsa-m

iR-2

22

hsa-m

iR-1

46a

hsa-m

iR-3

9cel-m

iR-3

9cel-m

iR-2

38

Regions

Ne

t S

ign

al in

ten

sit

y (

MF

I)

50ng

100ng

250ng

500ng








bull Flexible





Proteomic Assays



Nucleic Acids








or IgM





Multiplex Detection

Detection


IgG

1 2 3 4





















Y Y

Y

IgG1 IgG2

IgG3

IgG4 IgA



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Detection



Antigen

1 2 3








Y Y Y

Y

Y

Y Y

Y Y

Y Y

Y



Y


Detection

Multiplex Detection


















compounds





compounds



SAPE SAPE














SAPE SAPE


Detection

Multiplex Detection












543-550






GTPases 827 271-282














Single Plex Assays

To Luminex

Multiplex

Assays






1

2

TAG-1

TAG-2



o Gene expression



o miRNA analysis


applications




ANNEAL

Ligate

PCR

TAG1

TAG2

cDNA or

gDNAPCR

HYBRIDIZE

amp

LABEL

dA

dG dT

dC -

ANNEAL

amp

Extend

TAG1

TAG2

HYBRIDIZE

amp

LABEL

gDNAPCR

SNP typing CF

CYP 450 RVP


CNV etc

RVP FAST GPP FAST

HYBRIDIZE

amp

LABEL

TAG1

TAG2 ANNEAL

amp

PCR

gDNA


30



2 hours



30 minutes



30 minutes





30 minutes







methods







TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

AAAAAAA RNA

AAAAAAA RNA

AAAAAAA RNA






TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

AAAAAAA RNA

AAAAAAA RNA

AAAAAAA RNA

cDNA

cDNA

cDNA




TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

cDNA

cDNA

cDNA




LMA Probe Design



Upstream LMA Probe

cDNA


Universal Primer 1

5rsquo 3rsquo












TAG

SAPE

Detection



lt3 Hours Setup











ANNEAL

Ligate

PCR

TAG1

TAG2

cDNA or

gDNAPCR

HYBRIDIZE

amp

LABEL





Upstream LMA Probes

T

Universal Primer 1

5rsquo

A

5rsquo

G



SNP

T



T

T


PCR Amplification

UP 1

P

No Amplification

B-UP 2



Universal Prime mix

UP 1

P

B-UP 2

UP 1

P

B-UP 2

Ligate

T T







TAG

SAPE

Detection



Bead Hyb 80 minutes







Incubation








Targeted PCR




Upstream LDR Probes

A T

G



SNP

A

A



A


Multiple

Ligation

cycles









TAG

SAPE

Detection






Forward primers

gDNA


5rsquo

3rsquo


A

T

5rsquo

3rsquo

G





Targeted PCR



A

5rsquo

3rsquo

G

5rsquo

3rsquo

T




A

5rsquo

3rsquo

G 5rsquo

3rsquo

T








xTAG


Detection

SAPE










xTAG primer

gDNA


Biotinylated primer

5rsquo 3rsquo

3rsquo

dATP dTTP dGTP dCTP

= Carbon Spacer











Detection

Sequence Detection

TAG


















Workflow

75



2 hours



30 minutes



30 minutes





30 minutes

76






0

500

1000

1500

2000

2500

3000

3500

4000


Sample

Sig

nal In

ten

sit

y (

MF

I)

let-7c

Let-7a

77


miR-107 vs miR-103

0

1000

2000

3000

4000

5000

6000


Sample

Sig

na

l In

ten

sit

y (

MF

I)

miR-103

miR-107









0

1000

2000

3000

4000

5000

6000

7000

8000

9000

10000

hsa-m

iR-1

07

hsa-m

iR-1

47

hsa-m

iR-1

81a

hsa-m

iR-1

92

hsa-m

iR-1

9b

hsa-m

iR-2

20a

hsa-m

iR-2

1hsa-m

iR-2

18

hsa-le

t-7c

hsa-m

iR-1

00

hsa-m

iR-1

25b

hsa-m

iR-

hsa-m

iR-2

05

hsa-m

iR-2

21

hsa-m

iR-2

00a

hsa-m

iR-2

00c

hsa-m

iR-2

06

hsa-m

iR-1

0a

hsa-m

iR-1

6

hsa-m

iR-9

hsa-m

iR-4

24

hsa-m

iR-2

0a

hsa-m

iR-2

0b

hsa-le

t-7a

hsa-m

iR-9

5hsa-m

iR-1

36

hsa-m

iR-1

95

hsa-m

iR-2

10

hsa-m

iR-2

12

hsa-m

iR-

hsa-m

iR-1

7

hsa-m

iR-1

22

hsa-m

iR-1

43

hsa-m

iR-1

45

hsa-m

iR-1

06a

hsa-m

iR-

hsa-m

iR-1

0b

hsa-m

iR-1

hsa-m

iR-2

23

hsa-m

iR-1

81b

hsa-m

iR-1

55

hsa-le

t-7g

hsa-le

t-7i

hsa-m

iR-9

6

hsa-m

iR-1

37

hsa-m

iR-1

85

hsa-m

iR-1

38

hsa-m

iR-1

98

hsa-m

iR-1

33a

hsa-m

iR-1

33b

hsa-m

iR-1

5a

hsa-m

iR-3

4a

hsa-m

iR-2

9a

hsa-m

iR-2

9b

hsa-m

iR-1

03

hsa-m

iR-2

22

hsa-m

iR-1

46a

cel-m

iR-3

9cel-m

iR-5

4

cel-m

iR-2

38

Regions

Ne

t S

ign

al In

ten

sit

y (

MF

I)

106a

17


Total RNA Samples

53-Plex Assay

miR-9 miR-122 miR-1

0

5000

10000

15000

20000

25000

Samples

Ne

t S

ign

al In

ten

sit

y (

MF

I)

miR-9

miR-122

miR-1






289704

12431797

4050

7044

12899

18030

24973

23884

21852

19249

11326

6573

3852

2471

0

5000

10000

15000

20000

25000

30000


Input Amount

Ne

t S

ign

al In

ten

sit

y (

MF

I)

200 amol


81


0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000



Ne

t S

ign

al in

ten

sit

y (

MF

I)

82

miR-133a

000

020

040

060

080

100

120


Total RNA Samples

Rela

tive C

han

ge t

o H

eart

FlexmiR v2

qRT-PCR

Tissue

Re

lati

ve

Ch

an

ge


LNP-miRNA

qRT-PCR

83

miRNA

Re

lati

ve

Ch

an

ge



0

5

10

15

20

25


miRNA

Rela

tive C

han

ge

FlexmiR v2

qRT-PCR

LNP-miRNA

qPCR

84



0

5000

10000

15000

20000

25000

30000

hsa-m

iR-1

07

hsa-m

iR-1

47

hsa-m

iR-1

81a

hsa-m

iR-1

92

hsa-m

iR-1

9b

hsa-m

iR-2

20a

hsa-m

iR-2

1hsa-m

iR-2

18

hsa-le

t-7c

hsa-m

iR-1

00

hsa-m

iR-1

25b

hsa-m

iR-

hsa-m

iR-2

05

hsa-m

iR-2

21

hsa-m

iR-2

00a

hsa-m

iR-2

00c

hsa-m

iR-2

06

hsa-m

iR-1

0a

hsa-m

iR-1

6hsa-m

iR-9

hsa-m

iR-4

24

hsa-m

iR-2

0a

hsa-m

iR-2

0b

hsa-le

t-7a

hsa-m

iR-9

5hsa-m

iR-1

36

hsa-m

iR-1

95

hsa-m

iR-2

10

hsa-m

iR-2

12

hsa-m

iR-

hsa-m

iR-1

7hsa-m

iR-1

22

hsa-m

iR-1

43

hsa-m

iR-1

45

hsa-m

ir-1

06a

hsa-m

iR-

hsa-m

iR-1

0b

hsa-m

iR-1

hsa-m

iR-2

23

hsa-m

iR-1

81b

hsa-le

t-155

hsa-le

t-7g

hsa-m

iR-7

ihsa-m

iR-9

6hsa-m

iR-1

37

hsa-m

iR-1

85

hsa-m

iR-1

38

hsa-m

iR-1

33a

hsa-m

iR-1

33b

hsa-m

iR-1

5a

hsa-m

iR-3

4a

hsa-m

iR-2

9a

hsa-m

iR-2

9b

hsa-m

iR-1

03

hsa-m

iR-2

22

hsa-m

iR-1

46a

hsa-m

iR-3

9cel-m

iR-3

9cel-m

iR-2

38

Regions

Ne

t S

ign

al in

ten

sit

y (

MF

I)

50ng

100ng

250ng

500ng








bull Flexible





Proteomic Assays



Nucleic Acids








or IgM





Multiplex Detection

Detection


IgG

1 2 3 4





















Y Y

Y

IgG1 IgG2

IgG3

IgG4 IgA



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Detection



Antigen

1 2 3








Y Y Y

Y

Y

Y Y

Y Y

Y Y

Y



Y


Detection

Multiplex Detection


















compounds





compounds



SAPE SAPE














SAPE SAPE


Detection

Multiplex Detection












543-550






GTPases 827 271-282














Single Plex Assays

To Luminex

Multiplex

Assays





o Gene expression



o miRNA analysis


applications




ANNEAL

Ligate

PCR

TAG1

TAG2

cDNA or

gDNAPCR

HYBRIDIZE

amp

LABEL

dA

dG dT

dC -

ANNEAL

amp

Extend

TAG1

TAG2

HYBRIDIZE

amp

LABEL

gDNAPCR

SNP typing CF

CYP 450 RVP


CNV etc

RVP FAST GPP FAST

HYBRIDIZE

amp

LABEL

TAG1

TAG2 ANNEAL

amp

PCR

gDNA


30



2 hours



30 minutes



30 minutes





30 minutes







methods







TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

AAAAAAA RNA

AAAAAAA RNA

AAAAAAA RNA






TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

AAAAAAA RNA

AAAAAAA RNA

AAAAAAA RNA

cDNA

cDNA

cDNA




TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

cDNA

cDNA

cDNA




LMA Probe Design



Upstream LMA Probe

cDNA


Universal Primer 1

5rsquo 3rsquo












TAG

SAPE

Detection



lt3 Hours Setup











ANNEAL

Ligate

PCR

TAG1

TAG2

cDNA or

gDNAPCR

HYBRIDIZE

amp

LABEL





Upstream LMA Probes

T

Universal Primer 1

5rsquo

A

5rsquo

G



SNP

T



T

T


PCR Amplification

UP 1

P

No Amplification

B-UP 2



Universal Prime mix

UP 1

P

B-UP 2

UP 1

P

B-UP 2

Ligate

T T







TAG

SAPE

Detection



Bead Hyb 80 minutes







Incubation








Targeted PCR




Upstream LDR Probes

A T

G



SNP

A

A



A


Multiple

Ligation

cycles









TAG

SAPE

Detection






Forward primers

gDNA


5rsquo

3rsquo


A

T

5rsquo

3rsquo

G





Targeted PCR



A

5rsquo

3rsquo

G

5rsquo

3rsquo

T




A

5rsquo

3rsquo

G 5rsquo

3rsquo

T








xTAG


Detection

SAPE










xTAG primer

gDNA


Biotinylated primer

5rsquo 3rsquo

3rsquo

dATP dTTP dGTP dCTP

= Carbon Spacer











Detection

Sequence Detection

TAG


















Workflow

75



2 hours



30 minutes



30 minutes





30 minutes

76






0

500

1000

1500

2000

2500

3000

3500

4000


Sample

Sig

nal In

ten

sit

y (

MF

I)

let-7c

Let-7a

77


miR-107 vs miR-103

0

1000

2000

3000

4000

5000

6000


Sample

Sig

na

l In

ten

sit

y (

MF

I)

miR-103

miR-107









0

1000

2000

3000

4000

5000

6000

7000

8000

9000

10000

hsa-m

iR-1

07

hsa-m

iR-1

47

hsa-m

iR-1

81a

hsa-m

iR-1

92

hsa-m

iR-1

9b

hsa-m

iR-2

20a

hsa-m

iR-2

1hsa-m

iR-2

18

hsa-le

t-7c

hsa-m

iR-1

00

hsa-m

iR-1

25b

hsa-m

iR-

hsa-m

iR-2

05

hsa-m

iR-2

21

hsa-m

iR-2

00a

hsa-m

iR-2

00c

hsa-m

iR-2

06

hsa-m

iR-1

0a

hsa-m

iR-1

6

hsa-m

iR-9

hsa-m

iR-4

24

hsa-m

iR-2

0a

hsa-m

iR-2

0b

hsa-le

t-7a

hsa-m

iR-9

5hsa-m

iR-1

36

hsa-m

iR-1

95

hsa-m

iR-2

10

hsa-m

iR-2

12

hsa-m

iR-

hsa-m

iR-1

7

hsa-m

iR-1

22

hsa-m

iR-1

43

hsa-m

iR-1

45

hsa-m

iR-1

06a

hsa-m

iR-

hsa-m

iR-1

0b

hsa-m

iR-1

hsa-m

iR-2

23

hsa-m

iR-1

81b

hsa-m

iR-1

55

hsa-le

t-7g

hsa-le

t-7i

hsa-m

iR-9

6

hsa-m

iR-1

37

hsa-m

iR-1

85

hsa-m

iR-1

38

hsa-m

iR-1

98

hsa-m

iR-1

33a

hsa-m

iR-1

33b

hsa-m

iR-1

5a

hsa-m

iR-3

4a

hsa-m

iR-2

9a

hsa-m

iR-2

9b

hsa-m

iR-1

03

hsa-m

iR-2

22

hsa-m

iR-1

46a

cel-m

iR-3

9cel-m

iR-5

4

cel-m

iR-2

38

Regions

Ne

t S

ign

al In

ten

sit

y (

MF

I)

106a

17


Total RNA Samples

53-Plex Assay

miR-9 miR-122 miR-1

0

5000

10000

15000

20000

25000

Samples

Ne

t S

ign

al In

ten

sit

y (

MF

I)

miR-9

miR-122

miR-1






289704

12431797

4050

7044

12899

18030

24973

23884

21852

19249

11326

6573

3852

2471

0

5000

10000

15000

20000

25000

30000


Input Amount

Ne

t S

ign

al In

ten

sit

y (

MF

I)

200 amol


81


0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000



Ne

t S

ign

al in

ten

sit

y (

MF

I)

82

miR-133a

000

020

040

060

080

100

120


Total RNA Samples

Rela

tive C

han

ge t

o H

eart

FlexmiR v2

qRT-PCR

Tissue

Re

lati

ve

Ch

an

ge


LNP-miRNA

qRT-PCR

83

miRNA

Re

lati

ve

Ch

an

ge



0

5

10

15

20

25


miRNA

Rela

tive C

han

ge

FlexmiR v2

qRT-PCR

LNP-miRNA

qPCR

84



0

5000

10000

15000

20000

25000

30000

hsa-m

iR-1

07

hsa-m

iR-1

47

hsa-m

iR-1

81a

hsa-m

iR-1

92

hsa-m

iR-1

9b

hsa-m

iR-2

20a

hsa-m

iR-2

1hsa-m

iR-2

18

hsa-le

t-7c

hsa-m

iR-1

00

hsa-m

iR-1

25b

hsa-m

iR-

hsa-m

iR-2

05

hsa-m

iR-2

21

hsa-m

iR-2

00a

hsa-m

iR-2

00c

hsa-m

iR-2

06

hsa-m

iR-1

0a

hsa-m

iR-1

6hsa-m

iR-9

hsa-m

iR-4

24

hsa-m

iR-2

0a

hsa-m

iR-2

0b

hsa-le

t-7a

hsa-m

iR-9

5hsa-m

iR-1

36

hsa-m

iR-1

95

hsa-m

iR-2

10

hsa-m

iR-2

12

hsa-m

iR-

hsa-m

iR-1

7hsa-m

iR-1

22

hsa-m

iR-1

43

hsa-m

iR-1

45

hsa-m

ir-1

06a

hsa-m

iR-

hsa-m

iR-1

0b

hsa-m

iR-1

hsa-m

iR-2

23

hsa-m

iR-1

81b

hsa-le

t-155

hsa-le

t-7g

hsa-m

iR-7

ihsa-m

iR-9

6hsa-m

iR-1

37

hsa-m

iR-1

85

hsa-m

iR-1

38

hsa-m

iR-1

33a

hsa-m

iR-1

33b

hsa-m

iR-1

5a

hsa-m

iR-3

4a

hsa-m

iR-2

9a

hsa-m

iR-2

9b

hsa-m

iR-1

03

hsa-m

iR-2

22

hsa-m

iR-1

46a

hsa-m

iR-3

9cel-m

iR-3

9cel-m

iR-2

38

Regions

Ne

t S

ign

al in

ten

sit

y (

MF

I)

50ng

100ng

250ng

500ng








bull Flexible





Proteomic Assays



Nucleic Acids








or IgM





Multiplex Detection

Detection


IgG

1 2 3 4





















Y Y

Y

IgG1 IgG2

IgG3

IgG4 IgA



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Detection



Antigen

1 2 3








Y Y Y

Y

Y

Y Y

Y Y

Y Y

Y



Y


Detection

Multiplex Detection


















compounds





compounds



SAPE SAPE














SAPE SAPE


Detection

Multiplex Detection












543-550






GTPases 827 271-282














Single Plex Assays

To Luminex

Multiplex

Assays






ANNEAL

Ligate

PCR

TAG1

TAG2

cDNA or

gDNAPCR

HYBRIDIZE

amp

LABEL

dA

dG dT

dC -

ANNEAL

amp

Extend

TAG1

TAG2

HYBRIDIZE

amp

LABEL

gDNAPCR

SNP typing CF

CYP 450 RVP


CNV etc

RVP FAST GPP FAST

HYBRIDIZE

amp

LABEL

TAG1

TAG2 ANNEAL

amp

PCR

gDNA


30



2 hours



30 minutes



30 minutes





30 minutes







methods







TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

AAAAAAA RNA

AAAAAAA RNA

AAAAAAA RNA






TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

AAAAAAA RNA

AAAAAAA RNA

AAAAAAA RNA

cDNA

cDNA

cDNA




TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

cDNA

cDNA

cDNA




LMA Probe Design



Upstream LMA Probe

cDNA


Universal Primer 1

5rsquo 3rsquo












TAG

SAPE

Detection



lt3 Hours Setup











ANNEAL

Ligate

PCR

TAG1

TAG2

cDNA or

gDNAPCR

HYBRIDIZE

amp

LABEL





Upstream LMA Probes

T

Universal Primer 1

5rsquo

A

5rsquo

G



SNP

T



T

T


PCR Amplification

UP 1

P

No Amplification

B-UP 2



Universal Prime mix

UP 1

P

B-UP 2

UP 1

P

B-UP 2

Ligate

T T







TAG

SAPE

Detection



Bead Hyb 80 minutes







Incubation








Targeted PCR




Upstream LDR Probes

A T

G



SNP

A

A



A


Multiple

Ligation

cycles









TAG

SAPE

Detection






Forward primers

gDNA


5rsquo

3rsquo


A

T

5rsquo

3rsquo

G





Targeted PCR



A

5rsquo

3rsquo

G

5rsquo

3rsquo

T




A

5rsquo

3rsquo

G 5rsquo

3rsquo

T








xTAG


Detection

SAPE










xTAG primer

gDNA


Biotinylated primer

5rsquo 3rsquo

3rsquo

dATP dTTP dGTP dCTP

= Carbon Spacer











Detection

Sequence Detection

TAG


















Workflow

75



2 hours



30 minutes



30 minutes





30 minutes

76






0

500

1000

1500

2000

2500

3000

3500

4000


Sample

Sig

nal In

ten

sit

y (

MF

I)

let-7c

Let-7a

77


miR-107 vs miR-103

0

1000

2000

3000

4000

5000

6000


Sample

Sig

na

l In

ten

sit

y (

MF

I)

miR-103

miR-107









0

1000

2000

3000

4000

5000

6000

7000

8000

9000

10000

hsa-m

iR-1

07

hsa-m

iR-1

47

hsa-m

iR-1

81a

hsa-m

iR-1

92

hsa-m

iR-1

9b

hsa-m

iR-2

20a

hsa-m

iR-2

1hsa-m

iR-2

18

hsa-le

t-7c

hsa-m

iR-1

00

hsa-m

iR-1

25b

hsa-m

iR-

hsa-m

iR-2

05

hsa-m

iR-2

21

hsa-m

iR-2

00a

hsa-m

iR-2

00c

hsa-m

iR-2

06

hsa-m

iR-1

0a

hsa-m

iR-1

6

hsa-m

iR-9

hsa-m

iR-4

24

hsa-m

iR-2

0a

hsa-m

iR-2

0b

hsa-le

t-7a

hsa-m

iR-9

5hsa-m

iR-1

36

hsa-m

iR-1

95

hsa-m

iR-2

10

hsa-m

iR-2

12

hsa-m

iR-

hsa-m

iR-1

7

hsa-m

iR-1

22

hsa-m

iR-1

43

hsa-m

iR-1

45

hsa-m

iR-1

06a

hsa-m

iR-

hsa-m

iR-1

0b

hsa-m

iR-1

hsa-m

iR-2

23

hsa-m

iR-1

81b

hsa-m

iR-1

55

hsa-le

t-7g

hsa-le

t-7i

hsa-m

iR-9

6

hsa-m

iR-1

37

hsa-m

iR-1

85

hsa-m

iR-1

38

hsa-m

iR-1

98

hsa-m

iR-1

33a

hsa-m

iR-1

33b

hsa-m

iR-1

5a

hsa-m

iR-3

4a

hsa-m

iR-2

9a

hsa-m

iR-2

9b

hsa-m

iR-1

03

hsa-m

iR-2

22

hsa-m

iR-1

46a

cel-m

iR-3

9cel-m

iR-5

4

cel-m

iR-2

38

Regions

Ne

t S

ign

al In

ten

sit

y (

MF

I)

106a

17


Total RNA Samples

53-Plex Assay

miR-9 miR-122 miR-1

0

5000

10000

15000

20000

25000

Samples

Ne

t S

ign

al In

ten

sit

y (

MF

I)

miR-9

miR-122

miR-1






289704

12431797

4050

7044

12899

18030

24973

23884

21852

19249

11326

6573

3852

2471

0

5000

10000

15000

20000

25000

30000


Input Amount

Ne

t S

ign

al In

ten

sit

y (

MF

I)

200 amol


81


0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000



Ne

t S

ign

al in

ten

sit

y (

MF

I)

82

miR-133a

000

020

040

060

080

100

120


Total RNA Samples

Rela

tive C

han

ge t

o H

eart

FlexmiR v2

qRT-PCR

Tissue

Re

lati

ve

Ch

an

ge


LNP-miRNA

qRT-PCR

83

miRNA

Re

lati

ve

Ch

an

ge



0

5

10

15

20

25


miRNA

Rela

tive C

han

ge

FlexmiR v2

qRT-PCR

LNP-miRNA

qPCR

84



0

5000

10000

15000

20000

25000

30000

hsa-m

iR-1

07

hsa-m

iR-1

47

hsa-m

iR-1

81a

hsa-m

iR-1

92

hsa-m

iR-1

9b

hsa-m

iR-2

20a

hsa-m

iR-2

1hsa-m

iR-2

18

hsa-le

t-7c

hsa-m

iR-1

00

hsa-m

iR-1

25b

hsa-m

iR-

hsa-m

iR-2

05

hsa-m

iR-2

21

hsa-m

iR-2

00a

hsa-m

iR-2

00c

hsa-m

iR-2

06

hsa-m

iR-1

0a

hsa-m

iR-1

6hsa-m

iR-9

hsa-m

iR-4

24

hsa-m

iR-2

0a

hsa-m

iR-2

0b

hsa-le

t-7a

hsa-m

iR-9

5hsa-m

iR-1

36

hsa-m

iR-1

95

hsa-m

iR-2

10

hsa-m

iR-2

12

hsa-m

iR-

hsa-m

iR-1

7hsa-m

iR-1

22

hsa-m

iR-1

43

hsa-m

iR-1

45

hsa-m

ir-1

06a

hsa-m

iR-

hsa-m

iR-1

0b

hsa-m

iR-1

hsa-m

iR-2

23

hsa-m

iR-1

81b

hsa-le

t-155

hsa-le

t-7g

hsa-m

iR-7

ihsa-m

iR-9

6hsa-m

iR-1

37

hsa-m

iR-1

85

hsa-m

iR-1

38

hsa-m

iR-1

33a

hsa-m

iR-1

33b

hsa-m

iR-1

5a

hsa-m

iR-3

4a

hsa-m

iR-2

9a

hsa-m

iR-2

9b

hsa-m

iR-1

03

hsa-m

iR-2

22

hsa-m

iR-1

46a

hsa-m

iR-3

9cel-m

iR-3

9cel-m

iR-2

38

Regions

Ne

t S

ign

al in

ten

sit

y (

MF

I)

50ng

100ng

250ng

500ng








bull Flexible





Proteomic Assays



Nucleic Acids








or IgM





Multiplex Detection

Detection


IgG

1 2 3 4





















Y Y

Y

IgG1 IgG2

IgG3

IgG4 IgA



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Detection



Antigen

1 2 3








Y Y Y

Y

Y

Y Y

Y Y

Y Y

Y



Y


Detection

Multiplex Detection


















compounds





compounds



SAPE SAPE














SAPE SAPE


Detection

Multiplex Detection












543-550






GTPases 827 271-282














Single Plex Assays

To Luminex

Multiplex

Assays




30



2 hours



30 minutes



30 minutes





30 minutes







methods







TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

AAAAAAA RNA

AAAAAAA RNA

AAAAAAA RNA






TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

AAAAAAA RNA

AAAAAAA RNA

AAAAAAA RNA

cDNA

cDNA

cDNA




TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

cDNA

cDNA

cDNA




LMA Probe Design



Upstream LMA Probe

cDNA


Universal Primer 1

5rsquo 3rsquo












TAG

SAPE

Detection



lt3 Hours Setup











ANNEAL

Ligate

PCR

TAG1

TAG2

cDNA or

gDNAPCR

HYBRIDIZE

amp

LABEL





Upstream LMA Probes

T

Universal Primer 1

5rsquo

A

5rsquo

G



SNP

T



T

T


PCR Amplification

UP 1

P

No Amplification

B-UP 2



Universal Prime mix

UP 1

P

B-UP 2

UP 1

P

B-UP 2

Ligate

T T







TAG

SAPE

Detection



Bead Hyb 80 minutes







Incubation








Targeted PCR




Upstream LDR Probes

A T

G



SNP

A

A



A


Multiple

Ligation

cycles









TAG

SAPE

Detection






Forward primers

gDNA


5rsquo

3rsquo


A

T

5rsquo

3rsquo

G





Targeted PCR



A

5rsquo

3rsquo

G

5rsquo

3rsquo

T




A

5rsquo

3rsquo

G 5rsquo

3rsquo

T








xTAG


Detection

SAPE










xTAG primer

gDNA


Biotinylated primer

5rsquo 3rsquo

3rsquo

dATP dTTP dGTP dCTP

= Carbon Spacer











Detection

Sequence Detection

TAG


















Workflow

75



2 hours



30 minutes



30 minutes





30 minutes

76






0

500

1000

1500

2000

2500

3000

3500

4000


Sample

Sig

nal In

ten

sit

y (

MF

I)

let-7c

Let-7a

77


miR-107 vs miR-103

0

1000

2000

3000

4000

5000

6000


Sample

Sig

na

l In

ten

sit

y (

MF

I)

miR-103

miR-107









0

1000

2000

3000

4000

5000

6000

7000

8000

9000

10000

hsa-m

iR-1

07

hsa-m

iR-1

47

hsa-m

iR-1

81a

hsa-m

iR-1

92

hsa-m

iR-1

9b

hsa-m

iR-2

20a

hsa-m

iR-2

1hsa-m

iR-2

18

hsa-le

t-7c

hsa-m

iR-1

00

hsa-m

iR-1

25b

hsa-m

iR-

hsa-m

iR-2

05

hsa-m

iR-2

21

hsa-m

iR-2

00a

hsa-m

iR-2

00c

hsa-m

iR-2

06

hsa-m

iR-1

0a

hsa-m

iR-1

6

hsa-m

iR-9

hsa-m

iR-4

24

hsa-m

iR-2

0a

hsa-m

iR-2

0b

hsa-le

t-7a

hsa-m

iR-9

5hsa-m

iR-1

36

hsa-m

iR-1

95

hsa-m

iR-2

10

hsa-m

iR-2

12

hsa-m

iR-

hsa-m

iR-1

7

hsa-m

iR-1

22

hsa-m

iR-1

43

hsa-m

iR-1

45

hsa-m

iR-1

06a

hsa-m

iR-

hsa-m

iR-1

0b

hsa-m

iR-1

hsa-m

iR-2

23

hsa-m

iR-1

81b

hsa-m

iR-1

55

hsa-le

t-7g

hsa-le

t-7i

hsa-m

iR-9

6

hsa-m

iR-1

37

hsa-m

iR-1

85

hsa-m

iR-1

38

hsa-m

iR-1

98

hsa-m

iR-1

33a

hsa-m

iR-1

33b

hsa-m

iR-1

5a

hsa-m

iR-3

4a

hsa-m

iR-2

9a

hsa-m

iR-2

9b

hsa-m

iR-1

03

hsa-m

iR-2

22

hsa-m

iR-1

46a

cel-m

iR-3

9cel-m

iR-5

4

cel-m

iR-2

38

Regions

Ne

t S

ign

al In

ten

sit

y (

MF

I)

106a

17


Total RNA Samples

53-Plex Assay

miR-9 miR-122 miR-1

0

5000

10000

15000

20000

25000

Samples

Ne

t S

ign

al In

ten

sit

y (

MF

I)

miR-9

miR-122

miR-1






289704

12431797

4050

7044

12899

18030

24973

23884

21852

19249

11326

6573

3852

2471

0

5000

10000

15000

20000

25000

30000


Input Amount

Ne

t S

ign

al In

ten

sit

y (

MF

I)

200 amol


81


0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000



Ne

t S

ign

al in

ten

sit

y (

MF

I)

82

miR-133a

000

020

040

060

080

100

120


Total RNA Samples

Rela

tive C

han

ge t

o H

eart

FlexmiR v2

qRT-PCR

Tissue

Re

lati

ve

Ch

an

ge


LNP-miRNA

qRT-PCR

83

miRNA

Re

lati

ve

Ch

an

ge



0

5

10

15

20

25


miRNA

Rela

tive C

han

ge

FlexmiR v2

qRT-PCR

LNP-miRNA

qPCR

84



0

5000

10000

15000

20000

25000

30000

hsa-m

iR-1

07

hsa-m

iR-1

47

hsa-m

iR-1

81a

hsa-m

iR-1

92

hsa-m

iR-1

9b

hsa-m

iR-2

20a

hsa-m

iR-2

1hsa-m

iR-2

18

hsa-le

t-7c

hsa-m

iR-1

00

hsa-m

iR-1

25b

hsa-m

iR-

hsa-m

iR-2

05

hsa-m

iR-2

21

hsa-m

iR-2

00a

hsa-m

iR-2

00c

hsa-m

iR-2

06

hsa-m

iR-1

0a

hsa-m

iR-1

6hsa-m

iR-9

hsa-m

iR-4

24

hsa-m

iR-2

0a

hsa-m

iR-2

0b

hsa-le

t-7a

hsa-m

iR-9

5hsa-m

iR-1

36

hsa-m

iR-1

95

hsa-m

iR-2

10

hsa-m

iR-2

12

hsa-m

iR-

hsa-m

iR-1

7hsa-m

iR-1

22

hsa-m

iR-1

43

hsa-m

iR-1

45

hsa-m

ir-1

06a

hsa-m

iR-

hsa-m

iR-1

0b

hsa-m

iR-1

hsa-m

iR-2

23

hsa-m

iR-1

81b

hsa-le

t-155

hsa-le

t-7g

hsa-m

iR-7

ihsa-m

iR-9

6hsa-m

iR-1

37

hsa-m

iR-1

85

hsa-m

iR-1

38

hsa-m

iR-1

33a

hsa-m

iR-1

33b

hsa-m

iR-1

5a

hsa-m

iR-3

4a

hsa-m

iR-2

9a

hsa-m

iR-2

9b

hsa-m

iR-1

03

hsa-m

iR-2

22

hsa-m

iR-1

46a

hsa-m

iR-3

9cel-m

iR-3

9cel-m

iR-2

38

Regions

Ne

t S

ign

al in

ten

sit

y (

MF

I)

50ng

100ng

250ng

500ng








bull Flexible





Proteomic Assays



Nucleic Acids








or IgM





Multiplex Detection

Detection


IgG

1 2 3 4





















Y Y

Y

IgG1 IgG2

IgG3

IgG4 IgA



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Detection



Antigen

1 2 3








Y Y Y

Y

Y

Y Y

Y Y

Y Y

Y



Y


Detection

Multiplex Detection


















compounds





compounds



SAPE SAPE














SAPE SAPE


Detection

Multiplex Detection












543-550






GTPases 827 271-282














Single Plex Assays

To Luminex

Multiplex

Assays









methods







TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

AAAAAAA RNA

AAAAAAA RNA

AAAAAAA RNA






TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

AAAAAAA RNA

AAAAAAA RNA

AAAAAAA RNA

cDNA

cDNA

cDNA




TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

cDNA

cDNA

cDNA




LMA Probe Design



Upstream LMA Probe

cDNA


Universal Primer 1

5rsquo 3rsquo












TAG

SAPE

Detection



lt3 Hours Setup











ANNEAL

Ligate

PCR

TAG1

TAG2

cDNA or

gDNAPCR

HYBRIDIZE

amp

LABEL





Upstream LMA Probes

T

Universal Primer 1

5rsquo

A

5rsquo

G



SNP

T



T

T


PCR Amplification

UP 1

P

No Amplification

B-UP 2



Universal Prime mix

UP 1

P

B-UP 2

UP 1

P

B-UP 2

Ligate

T T







TAG

SAPE

Detection



Bead Hyb 80 minutes







Incubation








Targeted PCR




Upstream LDR Probes

A T

G



SNP

A

A



A


Multiple

Ligation

cycles









TAG

SAPE

Detection






Forward primers

gDNA


5rsquo

3rsquo


A

T

5rsquo

3rsquo

G





Targeted PCR



A

5rsquo

3rsquo

G

5rsquo

3rsquo

T




A

5rsquo

3rsquo

G 5rsquo

3rsquo

T








xTAG


Detection

SAPE










xTAG primer

gDNA


Biotinylated primer

5rsquo 3rsquo

3rsquo

dATP dTTP dGTP dCTP

= Carbon Spacer











Detection

Sequence Detection

TAG


















Workflow

75



2 hours



30 minutes



30 minutes





30 minutes

76






0

500

1000

1500

2000

2500

3000

3500

4000


Sample

Sig

nal In

ten

sit

y (

MF

I)

let-7c

Let-7a

77


miR-107 vs miR-103

0

1000

2000

3000

4000

5000

6000


Sample

Sig

na

l In

ten

sit

y (

MF

I)

miR-103

miR-107









0

1000

2000

3000

4000

5000

6000

7000

8000

9000

10000

hsa-m

iR-1

07

hsa-m

iR-1

47

hsa-m

iR-1

81a

hsa-m

iR-1

92

hsa-m

iR-1

9b

hsa-m

iR-2

20a

hsa-m

iR-2

1hsa-m

iR-2

18

hsa-le

t-7c

hsa-m

iR-1

00

hsa-m

iR-1

25b

hsa-m

iR-

hsa-m

iR-2

05

hsa-m

iR-2

21

hsa-m

iR-2

00a

hsa-m

iR-2

00c

hsa-m

iR-2

06

hsa-m

iR-1

0a

hsa-m

iR-1

6

hsa-m

iR-9

hsa-m

iR-4

24

hsa-m

iR-2

0a

hsa-m

iR-2

0b

hsa-le

t-7a

hsa-m

iR-9

5hsa-m

iR-1

36

hsa-m

iR-1

95

hsa-m

iR-2

10

hsa-m

iR-2

12

hsa-m

iR-

hsa-m

iR-1

7

hsa-m

iR-1

22

hsa-m

iR-1

43

hsa-m

iR-1

45

hsa-m

iR-1

06a

hsa-m

iR-

hsa-m

iR-1

0b

hsa-m

iR-1

hsa-m

iR-2

23

hsa-m

iR-1

81b

hsa-m

iR-1

55

hsa-le

t-7g

hsa-le

t-7i

hsa-m

iR-9

6

hsa-m

iR-1

37

hsa-m

iR-1

85

hsa-m

iR-1

38

hsa-m

iR-1

98

hsa-m

iR-1

33a

hsa-m

iR-1

33b

hsa-m

iR-1

5a

hsa-m

iR-3

4a

hsa-m

iR-2

9a

hsa-m

iR-2

9b

hsa-m

iR-1

03

hsa-m

iR-2

22

hsa-m

iR-1

46a

cel-m

iR-3

9cel-m

iR-5

4

cel-m

iR-2

38

Regions

Ne

t S

ign

al In

ten

sit

y (

MF

I)

106a

17


Total RNA Samples

53-Plex Assay

miR-9 miR-122 miR-1

0

5000

10000

15000

20000

25000

Samples

Ne

t S

ign

al In

ten

sit

y (

MF

I)

miR-9

miR-122

miR-1






289704

12431797

4050

7044

12899

18030

24973

23884

21852

19249

11326

6573

3852

2471

0

5000

10000

15000

20000

25000

30000


Input Amount

Ne

t S

ign

al In

ten

sit

y (

MF

I)

200 amol


81


0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000



Ne

t S

ign

al in

ten

sit

y (

MF

I)

82

miR-133a

000

020

040

060

080

100

120


Total RNA Samples

Rela

tive C

han

ge t

o H

eart

FlexmiR v2

qRT-PCR

Tissue

Re

lati

ve

Ch

an

ge


LNP-miRNA

qRT-PCR

83

miRNA

Re

lati

ve

Ch

an

ge



0

5

10

15

20

25


miRNA

Rela

tive C

han

ge

FlexmiR v2

qRT-PCR

LNP-miRNA

qPCR

84



0

5000

10000

15000

20000

25000

30000

hsa-m

iR-1

07

hsa-m

iR-1

47

hsa-m

iR-1

81a

hsa-m

iR-1

92

hsa-m

iR-1

9b

hsa-m

iR-2

20a

hsa-m

iR-2

1hsa-m

iR-2

18

hsa-le

t-7c

hsa-m

iR-1

00

hsa-m

iR-1

25b

hsa-m

iR-

hsa-m

iR-2

05

hsa-m

iR-2

21

hsa-m

iR-2

00a

hsa-m

iR-2

00c

hsa-m

iR-2

06

hsa-m

iR-1

0a

hsa-m

iR-1

6hsa-m

iR-9

hsa-m

iR-4

24

hsa-m

iR-2

0a

hsa-m

iR-2

0b

hsa-le

t-7a

hsa-m

iR-9

5hsa-m

iR-1

36

hsa-m

iR-1

95

hsa-m

iR-2

10

hsa-m

iR-2

12

hsa-m

iR-

hsa-m

iR-1

7hsa-m

iR-1

22

hsa-m

iR-1

43

hsa-m

iR-1

45

hsa-m

ir-1

06a

hsa-m

iR-

hsa-m

iR-1

0b

hsa-m

iR-1

hsa-m

iR-2

23

hsa-m

iR-1

81b

hsa-le

t-155

hsa-le

t-7g

hsa-m

iR-7

ihsa-m

iR-9

6hsa-m

iR-1

37

hsa-m

iR-1

85

hsa-m

iR-1

38

hsa-m

iR-1

33a

hsa-m

iR-1

33b

hsa-m

iR-1

5a

hsa-m

iR-3

4a

hsa-m

iR-2

9a

hsa-m

iR-2

9b

hsa-m

iR-1

03

hsa-m

iR-2

22

hsa-m

iR-1

46a

hsa-m

iR-3

9cel-m

iR-3

9cel-m

iR-2

38

Regions

Ne

t S

ign

al in

ten

sit

y (

MF

I)

50ng

100ng

250ng

500ng








bull Flexible





Proteomic Assays



Nucleic Acids








or IgM





Multiplex Detection

Detection


IgG

1 2 3 4





















Y Y

Y

IgG1 IgG2

IgG3

IgG4 IgA



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Detection



Antigen

1 2 3








Y Y Y

Y

Y

Y Y

Y Y

Y Y

Y



Y


Detection

Multiplex Detection


















compounds





compounds



SAPE SAPE














SAPE SAPE


Detection

Multiplex Detection












543-550






GTPases 827 271-282














Single Plex Assays

To Luminex

Multiplex

Assays








methods







TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

AAAAAAA RNA

AAAAAAA RNA

AAAAAAA RNA






TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

AAAAAAA RNA

AAAAAAA RNA

AAAAAAA RNA

cDNA

cDNA

cDNA




TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

cDNA

cDNA

cDNA




LMA Probe Design



Upstream LMA Probe

cDNA


Universal Primer 1

5rsquo 3rsquo












TAG

SAPE

Detection



lt3 Hours Setup











ANNEAL

Ligate

PCR

TAG1

TAG2

cDNA or

gDNAPCR

HYBRIDIZE

amp

LABEL





Upstream LMA Probes

T

Universal Primer 1

5rsquo

A

5rsquo

G



SNP

T



T

T


PCR Amplification

UP 1

P

No Amplification

B-UP 2



Universal Prime mix

UP 1

P

B-UP 2

UP 1

P

B-UP 2

Ligate

T T







TAG

SAPE

Detection



Bead Hyb 80 minutes







Incubation








Targeted PCR




Upstream LDR Probes

A T

G



SNP

A

A



A


Multiple

Ligation

cycles









TAG

SAPE

Detection






Forward primers

gDNA


5rsquo

3rsquo


A

T

5rsquo

3rsquo

G





Targeted PCR



A

5rsquo

3rsquo

G

5rsquo

3rsquo

T




A

5rsquo

3rsquo

G 5rsquo

3rsquo

T








xTAG


Detection

SAPE










xTAG primer

gDNA


Biotinylated primer

5rsquo 3rsquo

3rsquo

dATP dTTP dGTP dCTP

= Carbon Spacer











Detection

Sequence Detection

TAG


















Workflow

75



2 hours



30 minutes



30 minutes





30 minutes

76






0

500

1000

1500

2000

2500

3000

3500

4000


Sample

Sig

nal In

ten

sit

y (

MF

I)

let-7c

Let-7a

77


miR-107 vs miR-103

0

1000

2000

3000

4000

5000

6000


Sample

Sig

na

l In

ten

sit

y (

MF

I)

miR-103

miR-107









0

1000

2000

3000

4000

5000

6000

7000

8000

9000

10000

hsa-m

iR-1

07

hsa-m

iR-1

47

hsa-m

iR-1

81a

hsa-m

iR-1

92

hsa-m

iR-1

9b

hsa-m

iR-2

20a

hsa-m

iR-2

1hsa-m

iR-2

18

hsa-le

t-7c

hsa-m

iR-1

00

hsa-m

iR-1

25b

hsa-m

iR-

hsa-m

iR-2

05

hsa-m

iR-2

21

hsa-m

iR-2

00a

hsa-m

iR-2

00c

hsa-m

iR-2

06

hsa-m

iR-1

0a

hsa-m

iR-1

6

hsa-m

iR-9

hsa-m

iR-4

24

hsa-m

iR-2

0a

hsa-m

iR-2

0b

hsa-le

t-7a

hsa-m

iR-9

5hsa-m

iR-1

36

hsa-m

iR-1

95

hsa-m

iR-2

10

hsa-m

iR-2

12

hsa-m

iR-

hsa-m

iR-1

7

hsa-m

iR-1

22

hsa-m

iR-1

43

hsa-m

iR-1

45

hsa-m

iR-1

06a

hsa-m

iR-

hsa-m

iR-1

0b

hsa-m

iR-1

hsa-m

iR-2

23

hsa-m

iR-1

81b

hsa-m

iR-1

55

hsa-le

t-7g

hsa-le

t-7i

hsa-m

iR-9

6

hsa-m

iR-1

37

hsa-m

iR-1

85

hsa-m

iR-1

38

hsa-m

iR-1

98

hsa-m

iR-1

33a

hsa-m

iR-1

33b

hsa-m

iR-1

5a

hsa-m

iR-3

4a

hsa-m

iR-2

9a

hsa-m

iR-2

9b

hsa-m

iR-1

03

hsa-m

iR-2

22

hsa-m

iR-1

46a

cel-m

iR-3

9cel-m

iR-5

4

cel-m

iR-2

38

Regions

Ne

t S

ign

al In

ten

sit

y (

MF

I)

106a

17


Total RNA Samples

53-Plex Assay

miR-9 miR-122 miR-1

0

5000

10000

15000

20000

25000

Samples

Ne

t S

ign

al In

ten

sit

y (

MF

I)

miR-9

miR-122

miR-1






289704

12431797

4050

7044

12899

18030

24973

23884

21852

19249

11326

6573

3852

2471

0

5000

10000

15000

20000

25000

30000


Input Amount

Ne

t S

ign

al In

ten

sit

y (

MF

I)

200 amol


81


0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000



Ne

t S

ign

al in

ten

sit

y (

MF

I)

82

miR-133a

000

020

040

060

080

100

120


Total RNA Samples

Rela

tive C

han

ge t

o H

eart

FlexmiR v2

qRT-PCR

Tissue

Re

lati

ve

Ch

an

ge


LNP-miRNA

qRT-PCR

83

miRNA

Re

lati

ve

Ch

an

ge



0

5

10

15

20

25


miRNA

Rela

tive C

han

ge

FlexmiR v2

qRT-PCR

LNP-miRNA

qPCR

84



0

5000

10000

15000

20000

25000

30000

hsa-m

iR-1

07

hsa-m

iR-1

47

hsa-m

iR-1

81a

hsa-m

iR-1

92

hsa-m

iR-1

9b

hsa-m

iR-2

20a

hsa-m

iR-2

1hsa-m

iR-2

18

hsa-le

t-7c

hsa-m

iR-1

00

hsa-m

iR-1

25b

hsa-m

iR-

hsa-m

iR-2

05

hsa-m

iR-2

21

hsa-m

iR-2

00a

hsa-m

iR-2

00c

hsa-m

iR-2

06

hsa-m

iR-1

0a

hsa-m

iR-1

6hsa-m

iR-9

hsa-m

iR-4

24

hsa-m

iR-2

0a

hsa-m

iR-2

0b

hsa-le

t-7a

hsa-m

iR-9

5hsa-m

iR-1

36

hsa-m

iR-1

95

hsa-m

iR-2

10

hsa-m

iR-2

12

hsa-m

iR-

hsa-m

iR-1

7hsa-m

iR-1

22

hsa-m

iR-1

43

hsa-m

iR-1

45

hsa-m

ir-1

06a

hsa-m

iR-

hsa-m

iR-1

0b

hsa-m

iR-1

hsa-m

iR-2

23

hsa-m

iR-1

81b

hsa-le

t-155

hsa-le

t-7g

hsa-m

iR-7

ihsa-m

iR-9

6hsa-m

iR-1

37

hsa-m

iR-1

85

hsa-m

iR-1

38

hsa-m

iR-1

33a

hsa-m

iR-1

33b

hsa-m

iR-1

5a

hsa-m

iR-3

4a

hsa-m

iR-2

9a

hsa-m

iR-2

9b

hsa-m

iR-1

03

hsa-m

iR-2

22

hsa-m

iR-1

46a

hsa-m

iR-3

9cel-m

iR-3

9cel-m

iR-2

38

Regions

Ne

t S

ign

al in

ten

sit

y (

MF

I)

50ng

100ng

250ng

500ng








bull Flexible





Proteomic Assays



Nucleic Acids








or IgM





Multiplex Detection

Detection


IgG

1 2 3 4





















Y Y

Y

IgG1 IgG2

IgG3

IgG4 IgA



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Detection



Antigen

1 2 3








Y Y Y

Y

Y

Y Y

Y Y

Y Y

Y



Y


Detection

Multiplex Detection


















compounds





compounds



SAPE SAPE














SAPE SAPE


Detection

Multiplex Detection












543-550






GTPases 827 271-282














Single Plex Assays

To Luminex

Multiplex

Assays





TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

AAAAAAA RNA

AAAAAAA RNA

AAAAAAA RNA






TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

AAAAAAA RNA

AAAAAAA RNA

AAAAAAA RNA

cDNA

cDNA

cDNA




TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

cDNA

cDNA

cDNA




LMA Probe Design



Upstream LMA Probe

cDNA


Universal Primer 1

5rsquo 3rsquo












TAG

SAPE

Detection



lt3 Hours Setup











ANNEAL

Ligate

PCR

TAG1

TAG2

cDNA or

gDNAPCR

HYBRIDIZE

amp

LABEL





Upstream LMA Probes

T

Universal Primer 1

5rsquo

A

5rsquo

G



SNP

T



T

T


PCR Amplification

UP 1

P

No Amplification

B-UP 2



Universal Prime mix

UP 1

P

B-UP 2

UP 1

P

B-UP 2

Ligate

T T







TAG

SAPE

Detection



Bead Hyb 80 minutes







Incubation








Targeted PCR




Upstream LDR Probes

A T

G



SNP

A

A



A


Multiple

Ligation

cycles









TAG

SAPE

Detection






Forward primers

gDNA


5rsquo

3rsquo


A

T

5rsquo

3rsquo

G





Targeted PCR



A

5rsquo

3rsquo

G

5rsquo

3rsquo

T




A

5rsquo

3rsquo

G 5rsquo

3rsquo

T








xTAG


Detection

SAPE










xTAG primer

gDNA


Biotinylated primer

5rsquo 3rsquo

3rsquo

dATP dTTP dGTP dCTP

= Carbon Spacer











Detection

Sequence Detection

TAG


















Workflow

75



2 hours



30 minutes



30 minutes





30 minutes

76






0

500

1000

1500

2000

2500

3000

3500

4000


Sample

Sig

nal In

ten

sit

y (

MF

I)

let-7c

Let-7a

77


miR-107 vs miR-103

0

1000

2000

3000

4000

5000

6000


Sample

Sig

na

l In

ten

sit

y (

MF

I)

miR-103

miR-107









0

1000

2000

3000

4000

5000

6000

7000

8000

9000

10000

hsa-m

iR-1

07

hsa-m

iR-1

47

hsa-m

iR-1

81a

hsa-m

iR-1

92

hsa-m

iR-1

9b

hsa-m

iR-2

20a

hsa-m

iR-2

1hsa-m

iR-2

18

hsa-le

t-7c

hsa-m

iR-1

00

hsa-m

iR-1

25b

hsa-m

iR-

hsa-m

iR-2

05

hsa-m

iR-2

21

hsa-m

iR-2

00a

hsa-m

iR-2

00c

hsa-m

iR-2

06

hsa-m

iR-1

0a

hsa-m

iR-1

6

hsa-m

iR-9

hsa-m

iR-4

24

hsa-m

iR-2

0a

hsa-m

iR-2

0b

hsa-le

t-7a

hsa-m

iR-9

5hsa-m

iR-1

36

hsa-m

iR-1

95

hsa-m

iR-2

10

hsa-m

iR-2

12

hsa-m

iR-

hsa-m

iR-1

7

hsa-m

iR-1

22

hsa-m

iR-1

43

hsa-m

iR-1

45

hsa-m

iR-1

06a

hsa-m

iR-

hsa-m

iR-1

0b

hsa-m

iR-1

hsa-m

iR-2

23

hsa-m

iR-1

81b

hsa-m

iR-1

55

hsa-le

t-7g

hsa-le

t-7i

hsa-m

iR-9

6

hsa-m

iR-1

37

hsa-m

iR-1

85

hsa-m

iR-1

38

hsa-m

iR-1

98

hsa-m

iR-1

33a

hsa-m

iR-1

33b

hsa-m

iR-1

5a

hsa-m

iR-3

4a

hsa-m

iR-2

9a

hsa-m

iR-2

9b

hsa-m

iR-1

03

hsa-m

iR-2

22

hsa-m

iR-1

46a

cel-m

iR-3

9cel-m

iR-5

4

cel-m

iR-2

38

Regions

Ne

t S

ign

al In

ten

sit

y (

MF

I)

106a

17


Total RNA Samples

53-Plex Assay

miR-9 miR-122 miR-1

0

5000

10000

15000

20000

25000

Samples

Ne

t S

ign

al In

ten

sit

y (

MF

I)

miR-9

miR-122

miR-1






289704

12431797

4050

7044

12899

18030

24973

23884

21852

19249

11326

6573

3852

2471

0

5000

10000

15000

20000

25000

30000


Input Amount

Ne

t S

ign

al In

ten

sit

y (

MF

I)

200 amol


81


0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000



Ne

t S

ign

al in

ten

sit

y (

MF

I)

82

miR-133a

000

020

040

060

080

100

120


Total RNA Samples

Rela

tive C

han

ge t

o H

eart

FlexmiR v2

qRT-PCR

Tissue

Re

lati

ve

Ch

an

ge


LNP-miRNA

qRT-PCR

83

miRNA

Re

lati

ve

Ch

an

ge



0

5

10

15

20

25


miRNA

Rela

tive C

han

ge

FlexmiR v2

qRT-PCR

LNP-miRNA

qPCR

84



0

5000

10000

15000

20000

25000

30000

hsa-m

iR-1

07

hsa-m

iR-1

47

hsa-m

iR-1

81a

hsa-m

iR-1

92

hsa-m

iR-1

9b

hsa-m

iR-2

20a

hsa-m

iR-2

1hsa-m

iR-2

18

hsa-le

t-7c

hsa-m

iR-1

00

hsa-m

iR-1

25b

hsa-m

iR-

hsa-m

iR-2

05

hsa-m

iR-2

21

hsa-m

iR-2

00a

hsa-m

iR-2

00c

hsa-m

iR-2

06

hsa-m

iR-1

0a

hsa-m

iR-1

6hsa-m

iR-9

hsa-m

iR-4

24

hsa-m

iR-2

0a

hsa-m

iR-2

0b

hsa-le

t-7a

hsa-m

iR-9

5hsa-m

iR-1

36

hsa-m

iR-1

95

hsa-m

iR-2

10

hsa-m

iR-2

12

hsa-m

iR-

hsa-m

iR-1

7hsa-m

iR-1

22

hsa-m

iR-1

43

hsa-m

iR-1

45

hsa-m

ir-1

06a

hsa-m

iR-

hsa-m

iR-1

0b

hsa-m

iR-1

hsa-m

iR-2

23

hsa-m

iR-1

81b

hsa-le

t-155

hsa-le

t-7g

hsa-m

iR-7

ihsa-m

iR-9

6hsa-m

iR-1

37

hsa-m

iR-1

85

hsa-m

iR-1

38

hsa-m

iR-1

33a

hsa-m

iR-1

33b

hsa-m

iR-1

5a

hsa-m

iR-3

4a

hsa-m

iR-2

9a

hsa-m

iR-2

9b

hsa-m

iR-1

03

hsa-m

iR-2

22

hsa-m

iR-1

46a

hsa-m

iR-3

9cel-m

iR-3

9cel-m

iR-2

38

Regions

Ne

t S

ign

al in

ten

sit

y (

MF

I)

50ng

100ng

250ng

500ng








bull Flexible





Proteomic Assays



Nucleic Acids








or IgM





Multiplex Detection

Detection


IgG

1 2 3 4





















Y Y

Y

IgG1 IgG2

IgG3

IgG4 IgA



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Detection



Antigen

1 2 3








Y Y Y

Y

Y

Y Y

Y Y

Y Y

Y



Y


Detection

Multiplex Detection


















compounds





compounds



SAPE SAPE














SAPE SAPE


Detection

Multiplex Detection












543-550






GTPases 827 271-282














Single Plex Assays

To Luminex

Multiplex

Assays





TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

AAAAAAA RNA

AAAAAAA RNA

AAAAAAA RNA

cDNA

cDNA

cDNA




TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

cDNA

cDNA

cDNA




LMA Probe Design



Upstream LMA Probe

cDNA


Universal Primer 1

5rsquo 3rsquo












TAG

SAPE

Detection



lt3 Hours Setup











ANNEAL

Ligate

PCR

TAG1

TAG2

cDNA or

gDNAPCR

HYBRIDIZE

amp

LABEL





Upstream LMA Probes

T

Universal Primer 1

5rsquo

A

5rsquo

G



SNP

T



T

T


PCR Amplification

UP 1

P

No Amplification

B-UP 2



Universal Prime mix

UP 1

P

B-UP 2

UP 1

P

B-UP 2

Ligate

T T







TAG

SAPE

Detection



Bead Hyb 80 minutes







Incubation








Targeted PCR




Upstream LDR Probes

A T

G



SNP

A

A



A


Multiple

Ligation

cycles









TAG

SAPE

Detection






Forward primers

gDNA


5rsquo

3rsquo


A

T

5rsquo

3rsquo

G





Targeted PCR



A

5rsquo

3rsquo

G

5rsquo

3rsquo

T




A

5rsquo

3rsquo

G 5rsquo

3rsquo

T








xTAG


Detection

SAPE










xTAG primer

gDNA


Biotinylated primer

5rsquo 3rsquo

3rsquo

dATP dTTP dGTP dCTP

= Carbon Spacer











Detection

Sequence Detection

TAG


















Workflow

75



2 hours



30 minutes



30 minutes





30 minutes

76






0

500

1000

1500

2000

2500

3000

3500

4000


Sample

Sig

nal In

ten

sit

y (

MF

I)

let-7c

Let-7a

77


miR-107 vs miR-103

0

1000

2000

3000

4000

5000

6000


Sample

Sig

na

l In

ten

sit

y (

MF

I)

miR-103

miR-107









0

1000

2000

3000

4000

5000

6000

7000

8000

9000

10000

hsa-m

iR-1

07

hsa-m

iR-1

47

hsa-m

iR-1

81a

hsa-m

iR-1

92

hsa-m

iR-1

9b

hsa-m

iR-2

20a

hsa-m

iR-2

1hsa-m

iR-2

18

hsa-le

t-7c

hsa-m

iR-1

00

hsa-m

iR-1

25b

hsa-m

iR-

hsa-m

iR-2

05

hsa-m

iR-2

21

hsa-m

iR-2

00a

hsa-m

iR-2

00c

hsa-m

iR-2

06

hsa-m

iR-1

0a

hsa-m

iR-1

6

hsa-m

iR-9

hsa-m

iR-4

24

hsa-m

iR-2

0a

hsa-m

iR-2

0b

hsa-le

t-7a

hsa-m

iR-9

5hsa-m

iR-1

36

hsa-m

iR-1

95

hsa-m

iR-2

10

hsa-m

iR-2

12

hsa-m

iR-

hsa-m

iR-1

7

hsa-m

iR-1

22

hsa-m

iR-1

43

hsa-m

iR-1

45

hsa-m

iR-1

06a

hsa-m

iR-

hsa-m

iR-1

0b

hsa-m

iR-1

hsa-m

iR-2

23

hsa-m

iR-1

81b

hsa-m

iR-1

55

hsa-le

t-7g

hsa-le

t-7i

hsa-m

iR-9

6

hsa-m

iR-1

37

hsa-m

iR-1

85

hsa-m

iR-1

38

hsa-m

iR-1

98

hsa-m

iR-1

33a

hsa-m

iR-1

33b

hsa-m

iR-1

5a

hsa-m

iR-3

4a

hsa-m

iR-2

9a

hsa-m

iR-2

9b

hsa-m

iR-1

03

hsa-m

iR-2

22

hsa-m

iR-1

46a

cel-m

iR-3

9cel-m

iR-5

4

cel-m

iR-2

38

Regions

Ne

t S

ign

al In

ten

sit

y (

MF

I)

106a

17


Total RNA Samples

53-Plex Assay

miR-9 miR-122 miR-1

0

5000

10000

15000

20000

25000

Samples

Ne

t S

ign

al In

ten

sit

y (

MF

I)

miR-9

miR-122

miR-1






289704

12431797

4050

7044

12899

18030

24973

23884

21852

19249

11326

6573

3852

2471

0

5000

10000

15000

20000

25000

30000


Input Amount

Ne

t S

ign

al In

ten

sit

y (

MF

I)

200 amol


81


0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000



Ne

t S

ign

al in

ten

sit

y (

MF

I)

82

miR-133a

000

020

040

060

080

100

120


Total RNA Samples

Rela

tive C

han

ge t

o H

eart

FlexmiR v2

qRT-PCR

Tissue

Re

lati

ve

Ch

an

ge


LNP-miRNA

qRT-PCR

83

miRNA

Re

lati

ve

Ch

an

ge



0

5

10

15

20

25


miRNA

Rela

tive C

han

ge

FlexmiR v2

qRT-PCR

LNP-miRNA

qPCR

84



0

5000

10000

15000

20000

25000

30000

hsa-m

iR-1

07

hsa-m

iR-1

47

hsa-m

iR-1

81a

hsa-m

iR-1

92

hsa-m

iR-1

9b

hsa-m

iR-2

20a

hsa-m

iR-2

1hsa-m

iR-2

18

hsa-le

t-7c

hsa-m

iR-1

00

hsa-m

iR-1

25b

hsa-m

iR-

hsa-m

iR-2

05

hsa-m

iR-2

21

hsa-m

iR-2

00a

hsa-m

iR-2

00c

hsa-m

iR-2

06

hsa-m

iR-1

0a

hsa-m

iR-1

6hsa-m

iR-9

hsa-m

iR-4

24

hsa-m

iR-2

0a

hsa-m

iR-2

0b

hsa-le

t-7a

hsa-m

iR-9

5hsa-m

iR-1

36

hsa-m

iR-1

95

hsa-m

iR-2

10

hsa-m

iR-2

12

hsa-m

iR-

hsa-m

iR-1

7hsa-m

iR-1

22

hsa-m

iR-1

43

hsa-m

iR-1

45

hsa-m

ir-1

06a

hsa-m

iR-

hsa-m

iR-1

0b

hsa-m

iR-1

hsa-m

iR-2

23

hsa-m

iR-1

81b

hsa-le

t-155

hsa-le

t-7g

hsa-m

iR-7

ihsa-m

iR-9

6hsa-m

iR-1

37

hsa-m

iR-1

85

hsa-m

iR-1

38

hsa-m

iR-1

33a

hsa-m

iR-1

33b

hsa-m

iR-1

5a

hsa-m

iR-3

4a

hsa-m

iR-2

9a

hsa-m

iR-2

9b

hsa-m

iR-1

03

hsa-m

iR-2

22

hsa-m

iR-1

46a

hsa-m

iR-3

9cel-m

iR-3

9cel-m

iR-2

38

Regions

Ne

t S

ign

al in

ten

sit

y (

MF

I)

50ng

100ng

250ng

500ng








bull Flexible





Proteomic Assays



Nucleic Acids








or IgM





Multiplex Detection

Detection


IgG

1 2 3 4





















Y Y

Y

IgG1 IgG2

IgG3

IgG4 IgA



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Detection



Antigen

1 2 3








Y Y Y

Y

Y

Y Y

Y Y

Y Y

Y



Y


Detection

Multiplex Detection


















compounds





compounds



SAPE SAPE














SAPE SAPE


Detection

Multiplex Detection












543-550






GTPases 827 271-282














Single Plex Assays

To Luminex

Multiplex

Assays





TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

cDNA

cDNA

cDNA




LMA Probe Design



Upstream LMA Probe

cDNA


Universal Primer 1

5rsquo 3rsquo












TAG

SAPE

Detection



lt3 Hours Setup











ANNEAL

Ligate

PCR

TAG1

TAG2

cDNA or

gDNAPCR

HYBRIDIZE

amp

LABEL





Upstream LMA Probes

T

Universal Primer 1

5rsquo

A

5rsquo

G



SNP

T



T

T


PCR Amplification

UP 1

P

No Amplification

B-UP 2



Universal Prime mix

UP 1

P

B-UP 2

UP 1

P

B-UP 2

Ligate

T T







TAG

SAPE

Detection



Bead Hyb 80 minutes







Incubation








Targeted PCR




Upstream LDR Probes

A T

G



SNP

A

A



A


Multiple

Ligation

cycles









TAG

SAPE

Detection






Forward primers

gDNA


5rsquo

3rsquo


A

T

5rsquo

3rsquo

G





Targeted PCR



A

5rsquo

3rsquo

G

5rsquo

3rsquo

T




A

5rsquo

3rsquo

G 5rsquo

3rsquo

T








xTAG


Detection

SAPE










xTAG primer

gDNA


Biotinylated primer

5rsquo 3rsquo

3rsquo

dATP dTTP dGTP dCTP

= Carbon Spacer











Detection

Sequence Detection

TAG


















Workflow

75



2 hours



30 minutes



30 minutes





30 minutes

76






0

500

1000

1500

2000

2500

3000

3500

4000


Sample

Sig

nal In

ten

sit

y (

MF

I)

let-7c

Let-7a

77


miR-107 vs miR-103

0

1000

2000

3000

4000

5000

6000


Sample

Sig

na

l In

ten

sit

y (

MF

I)

miR-103

miR-107









0

1000

2000

3000

4000

5000

6000

7000

8000

9000

10000

hsa-m

iR-1

07

hsa-m

iR-1

47

hsa-m

iR-1

81a

hsa-m

iR-1

92

hsa-m

iR-1

9b

hsa-m

iR-2

20a

hsa-m

iR-2

1hsa-m

iR-2

18

hsa-le

t-7c

hsa-m

iR-1

00

hsa-m

iR-1

25b

hsa-m

iR-

hsa-m

iR-2

05

hsa-m

iR-2

21

hsa-m

iR-2

00a

hsa-m

iR-2

00c

hsa-m

iR-2

06

hsa-m

iR-1

0a

hsa-m

iR-1

6

hsa-m

iR-9

hsa-m

iR-4

24

hsa-m

iR-2

0a

hsa-m

iR-2

0b

hsa-le

t-7a

hsa-m

iR-9

5hsa-m

iR-1

36

hsa-m

iR-1

95

hsa-m

iR-2

10

hsa-m

iR-2

12

hsa-m

iR-

hsa-m

iR-1

7

hsa-m

iR-1

22

hsa-m

iR-1

43

hsa-m

iR-1

45

hsa-m

iR-1

06a

hsa-m

iR-

hsa-m

iR-1

0b

hsa-m

iR-1

hsa-m

iR-2

23

hsa-m

iR-1

81b

hsa-m

iR-1

55

hsa-le

t-7g

hsa-le

t-7i

hsa-m

iR-9

6

hsa-m

iR-1

37

hsa-m

iR-1

85

hsa-m

iR-1

38

hsa-m

iR-1

98

hsa-m

iR-1

33a

hsa-m

iR-1

33b

hsa-m

iR-1

5a

hsa-m

iR-3

4a

hsa-m

iR-2

9a

hsa-m

iR-2

9b

hsa-m

iR-1

03

hsa-m

iR-2

22

hsa-m

iR-1

46a

cel-m

iR-3

9cel-m

iR-5

4

cel-m

iR-2

38

Regions

Ne

t S

ign

al In

ten

sit

y (

MF

I)

106a

17


Total RNA Samples

53-Plex Assay

miR-9 miR-122 miR-1

0

5000

10000

15000

20000

25000

Samples

Ne

t S

ign

al In

ten

sit

y (

MF

I)

miR-9

miR-122

miR-1






289704

12431797

4050

7044

12899

18030

24973

23884

21852

19249

11326

6573

3852

2471

0

5000

10000

15000

20000

25000

30000


Input Amount

Ne

t S

ign

al In

ten

sit

y (

MF

I)

200 amol


81


0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000



Ne

t S

ign

al in

ten

sit

y (

MF

I)

82

miR-133a

000

020

040

060

080

100

120


Total RNA Samples

Rela

tive C

han

ge t

o H

eart

FlexmiR v2

qRT-PCR

Tissue

Re

lati

ve

Ch

an

ge


LNP-miRNA

qRT-PCR

83

miRNA

Re

lati

ve

Ch

an

ge



0

5

10

15

20

25


miRNA

Rela

tive C

han

ge

FlexmiR v2

qRT-PCR

LNP-miRNA

qPCR

84



0

5000

10000

15000

20000

25000

30000

hsa-m

iR-1

07

hsa-m

iR-1

47

hsa-m

iR-1

81a

hsa-m

iR-1

92

hsa-m

iR-1

9b

hsa-m

iR-2

20a

hsa-m

iR-2

1hsa-m

iR-2

18

hsa-le

t-7c

hsa-m

iR-1

00

hsa-m

iR-1

25b

hsa-m

iR-

hsa-m

iR-2

05

hsa-m

iR-2

21

hsa-m

iR-2

00a

hsa-m

iR-2

00c

hsa-m

iR-2

06

hsa-m

iR-1

0a

hsa-m

iR-1

6hsa-m

iR-9

hsa-m

iR-4

24

hsa-m

iR-2

0a

hsa-m

iR-2

0b

hsa-le

t-7a

hsa-m

iR-9

5hsa-m

iR-1

36

hsa-m

iR-1

95

hsa-m

iR-2

10

hsa-m

iR-2

12

hsa-m

iR-

hsa-m

iR-1

7hsa-m

iR-1

22

hsa-m

iR-1

43

hsa-m

iR-1

45

hsa-m

ir-1

06a

hsa-m

iR-

hsa-m

iR-1

0b

hsa-m

iR-1

hsa-m

iR-2

23

hsa-m

iR-1

81b

hsa-le

t-155

hsa-le

t-7g

hsa-m

iR-7

ihsa-m

iR-9

6hsa-m

iR-1

37

hsa-m

iR-1

85

hsa-m

iR-1

38

hsa-m

iR-1

33a

hsa-m

iR-1

33b

hsa-m

iR-1

5a

hsa-m

iR-3

4a

hsa-m

iR-2

9a

hsa-m

iR-2

9b

hsa-m

iR-1

03

hsa-m

iR-2

22

hsa-m

iR-1

46a

hsa-m

iR-3

9cel-m

iR-3

9cel-m

iR-2

38

Regions

Ne

t S

ign

al in

ten

sit

y (

MF

I)

50ng

100ng

250ng

500ng








bull Flexible





Proteomic Assays



Nucleic Acids








or IgM





Multiplex Detection

Detection


IgG

1 2 3 4





















Y Y

Y

IgG1 IgG2

IgG3

IgG4 IgA



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Detection



Antigen

1 2 3








Y Y Y

Y

Y

Y Y

Y Y

Y Y

Y



Y


Detection

Multiplex Detection


















compounds





compounds



SAPE SAPE














SAPE SAPE


Detection

Multiplex Detection












543-550






GTPases 827 271-282














Single Plex Assays

To Luminex

Multiplex

Assays



LMA Probe Design



Upstream LMA Probe

cDNA


Universal Primer 1

5rsquo 3rsquo












TAG

SAPE

Detection



lt3 Hours Setup











ANNEAL

Ligate

PCR

TAG1

TAG2

cDNA or

gDNAPCR

HYBRIDIZE

amp

LABEL





Upstream LMA Probes

T

Universal Primer 1

5rsquo

A

5rsquo

G



SNP

T



T

T


PCR Amplification

UP 1

P

No Amplification

B-UP 2



Universal Prime mix

UP 1

P

B-UP 2

UP 1

P

B-UP 2

Ligate

T T







TAG

SAPE

Detection



Bead Hyb 80 minutes







Incubation








Targeted PCR




Upstream LDR Probes

A T

G



SNP

A

A



A


Multiple

Ligation

cycles









TAG

SAPE

Detection






Forward primers

gDNA


5rsquo

3rsquo


A

T

5rsquo

3rsquo

G





Targeted PCR



A

5rsquo

3rsquo

G

5rsquo

3rsquo

T




A

5rsquo

3rsquo

G 5rsquo

3rsquo

T








xTAG


Detection

SAPE










xTAG primer

gDNA


Biotinylated primer

5rsquo 3rsquo

3rsquo

dATP dTTP dGTP dCTP

= Carbon Spacer











Detection

Sequence Detection

TAG


















Workflow

75



2 hours



30 minutes



30 minutes





30 minutes

76






0

500

1000

1500

2000

2500

3000

3500

4000


Sample

Sig

nal In

ten

sit

y (

MF

I)

let-7c

Let-7a

77


miR-107 vs miR-103

0

1000

2000

3000

4000

5000

6000


Sample

Sig

na

l In

ten

sit

y (

MF

I)

miR-103

miR-107









0

1000

2000

3000

4000

5000

6000

7000

8000

9000

10000

hsa-m

iR-1

07

hsa-m

iR-1

47

hsa-m

iR-1

81a

hsa-m

iR-1

92

hsa-m

iR-1

9b

hsa-m

iR-2

20a

hsa-m

iR-2

1hsa-m

iR-2

18

hsa-le

t-7c

hsa-m

iR-1

00

hsa-m

iR-1

25b

hsa-m

iR-

hsa-m

iR-2

05

hsa-m

iR-2

21

hsa-m

iR-2

00a

hsa-m

iR-2

00c

hsa-m

iR-2

06

hsa-m

iR-1

0a

hsa-m

iR-1

6

hsa-m

iR-9

hsa-m

iR-4

24

hsa-m

iR-2

0a

hsa-m

iR-2

0b

hsa-le

t-7a

hsa-m

iR-9

5hsa-m

iR-1

36

hsa-m

iR-1

95

hsa-m

iR-2

10

hsa-m

iR-2

12

hsa-m

iR-

hsa-m

iR-1

7

hsa-m

iR-1

22

hsa-m

iR-1

43

hsa-m

iR-1

45

hsa-m

iR-1

06a

hsa-m

iR-

hsa-m

iR-1

0b

hsa-m

iR-1

hsa-m

iR-2

23

hsa-m

iR-1

81b

hsa-m

iR-1

55

hsa-le

t-7g

hsa-le

t-7i

hsa-m

iR-9

6

hsa-m

iR-1

37

hsa-m

iR-1

85

hsa-m

iR-1

38

hsa-m

iR-1

98

hsa-m

iR-1

33a

hsa-m

iR-1

33b

hsa-m

iR-1

5a

hsa-m

iR-3

4a

hsa-m

iR-2

9a

hsa-m

iR-2

9b

hsa-m

iR-1

03

hsa-m

iR-2

22

hsa-m

iR-1

46a

cel-m

iR-3

9cel-m

iR-5

4

cel-m

iR-2

38

Regions

Ne

t S

ign

al In

ten

sit

y (

MF

I)

106a

17


Total RNA Samples

53-Plex Assay

miR-9 miR-122 miR-1

0

5000

10000

15000

20000

25000

Samples

Ne

t S

ign

al In

ten

sit

y (

MF

I)

miR-9

miR-122

miR-1






289704

12431797

4050

7044

12899

18030

24973

23884

21852

19249

11326

6573

3852

2471

0

5000

10000

15000

20000

25000

30000


Input Amount

Ne

t S

ign

al In

ten

sit

y (

MF

I)

200 amol


81


0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000



Ne

t S

ign

al in

ten

sit

y (

MF

I)

82

miR-133a

000

020

040

060

080

100

120


Total RNA Samples

Rela

tive C

han

ge t

o H

eart

FlexmiR v2

qRT-PCR

Tissue

Re

lati

ve

Ch

an

ge


LNP-miRNA

qRT-PCR

83

miRNA

Re

lati

ve

Ch

an

ge



0

5

10

15

20

25


miRNA

Rela

tive C

han

ge

FlexmiR v2

qRT-PCR

LNP-miRNA

qPCR

84



0

5000

10000

15000

20000

25000

30000

hsa-m

iR-1

07

hsa-m

iR-1

47

hsa-m

iR-1

81a

hsa-m

iR-1

92

hsa-m

iR-1

9b

hsa-m

iR-2

20a

hsa-m

iR-2

1hsa-m

iR-2

18

hsa-le

t-7c

hsa-m

iR-1

00

hsa-m

iR-1

25b

hsa-m

iR-

hsa-m

iR-2

05

hsa-m

iR-2

21

hsa-m

iR-2

00a

hsa-m

iR-2

00c

hsa-m

iR-2

06

hsa-m

iR-1

0a

hsa-m

iR-1

6hsa-m

iR-9

hsa-m

iR-4

24

hsa-m

iR-2

0a

hsa-m

iR-2

0b

hsa-le

t-7a

hsa-m

iR-9

5hsa-m

iR-1

36

hsa-m

iR-1

95

hsa-m

iR-2

10

hsa-m

iR-2

12

hsa-m

iR-

hsa-m

iR-1

7hsa-m

iR-1

22

hsa-m

iR-1

43

hsa-m

iR-1

45

hsa-m

ir-1

06a

hsa-m

iR-

hsa-m

iR-1

0b

hsa-m

iR-1

hsa-m

iR-2

23

hsa-m

iR-1

81b

hsa-le

t-155

hsa-le

t-7g

hsa-m

iR-7

ihsa-m

iR-9

6hsa-m

iR-1

37

hsa-m

iR-1

85

hsa-m

iR-1

38

hsa-m

iR-1

33a

hsa-m

iR-1

33b

hsa-m

iR-1

5a

hsa-m

iR-3

4a

hsa-m

iR-2

9a

hsa-m

iR-2

9b

hsa-m

iR-1

03

hsa-m

iR-2

22

hsa-m

iR-1

46a

hsa-m

iR-3

9cel-m

iR-3

9cel-m

iR-2

38

Regions

Ne

t S

ign

al in

ten

sit

y (

MF

I)

50ng

100ng

250ng

500ng








bull Flexible





Proteomic Assays



Nucleic Acids








or IgM





Multiplex Detection

Detection


IgG

1 2 3 4





















Y Y

Y

IgG1 IgG2

IgG3

IgG4 IgA



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Detection



Antigen

1 2 3








Y Y Y

Y

Y

Y Y

Y Y

Y Y

Y



Y


Detection

Multiplex Detection


















compounds





compounds



SAPE SAPE














SAPE SAPE


Detection

Multiplex Detection












543-550






GTPases 827 271-282














Single Plex Assays

To Luminex

Multiplex

Assays














TAG

SAPE

Detection



lt3 Hours Setup











ANNEAL

Ligate

PCR

TAG1

TAG2

cDNA or

gDNAPCR

HYBRIDIZE

amp

LABEL





Upstream LMA Probes

T

Universal Primer 1

5rsquo

A

5rsquo

G



SNP

T



T

T


PCR Amplification

UP 1

P

No Amplification

B-UP 2



Universal Prime mix

UP 1

P

B-UP 2

UP 1

P

B-UP 2

Ligate

T T







TAG

SAPE

Detection



Bead Hyb 80 minutes







Incubation








Targeted PCR




Upstream LDR Probes

A T

G



SNP

A

A



A


Multiple

Ligation

cycles









TAG

SAPE

Detection






Forward primers

gDNA


5rsquo

3rsquo


A

T

5rsquo

3rsquo

G





Targeted PCR



A

5rsquo

3rsquo

G

5rsquo

3rsquo

T




A

5rsquo

3rsquo

G 5rsquo

3rsquo

T








xTAG


Detection

SAPE










xTAG primer

gDNA


Biotinylated primer

5rsquo 3rsquo

3rsquo

dATP dTTP dGTP dCTP

= Carbon Spacer











Detection

Sequence Detection

TAG


















Workflow

75



2 hours



30 minutes



30 minutes





30 minutes

76






0

500

1000

1500

2000

2500

3000

3500

4000


Sample

Sig

nal In

ten

sit

y (

MF

I)

let-7c

Let-7a

77


miR-107 vs miR-103

0

1000

2000

3000

4000

5000

6000


Sample

Sig

na

l In

ten

sit

y (

MF

I)

miR-103

miR-107









0

1000

2000

3000

4000

5000

6000

7000

8000

9000

10000

hsa-m

iR-1

07

hsa-m

iR-1

47

hsa-m

iR-1

81a

hsa-m

iR-1

92

hsa-m

iR-1

9b

hsa-m

iR-2

20a

hsa-m

iR-2

1hsa-m

iR-2

18

hsa-le

t-7c

hsa-m

iR-1

00

hsa-m

iR-1

25b

hsa-m

iR-

hsa-m

iR-2

05

hsa-m

iR-2

21

hsa-m

iR-2

00a

hsa-m

iR-2

00c

hsa-m

iR-2

06

hsa-m

iR-1

0a

hsa-m

iR-1

6

hsa-m

iR-9

hsa-m

iR-4

24

hsa-m

iR-2

0a

hsa-m

iR-2

0b

hsa-le

t-7a

hsa-m

iR-9

5hsa-m

iR-1

36

hsa-m

iR-1

95

hsa-m

iR-2

10

hsa-m

iR-2

12

hsa-m

iR-

hsa-m

iR-1

7

hsa-m

iR-1

22

hsa-m

iR-1

43

hsa-m

iR-1

45

hsa-m

iR-1

06a

hsa-m

iR-

hsa-m

iR-1

0b

hsa-m

iR-1

hsa-m

iR-2

23

hsa-m

iR-1

81b

hsa-m

iR-1

55

hsa-le

t-7g

hsa-le

t-7i

hsa-m

iR-9

6

hsa-m

iR-1

37

hsa-m

iR-1

85

hsa-m

iR-1

38

hsa-m

iR-1

98

hsa-m

iR-1

33a

hsa-m

iR-1

33b

hsa-m

iR-1

5a

hsa-m

iR-3

4a

hsa-m

iR-2

9a

hsa-m

iR-2

9b

hsa-m

iR-1

03

hsa-m

iR-2

22

hsa-m

iR-1

46a

cel-m

iR-3

9cel-m

iR-5

4

cel-m

iR-2

38

Regions

Ne

t S

ign

al In

ten

sit

y (

MF

I)

106a

17


Total RNA Samples

53-Plex Assay

miR-9 miR-122 miR-1

0

5000

10000

15000

20000

25000

Samples

Ne

t S

ign

al In

ten

sit

y (

MF

I)

miR-9

miR-122

miR-1






289704

12431797

4050

7044

12899

18030

24973

23884

21852

19249

11326

6573

3852

2471

0

5000

10000

15000

20000

25000

30000


Input Amount

Ne

t S

ign

al In

ten

sit

y (

MF

I)

200 amol


81


0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000



Ne

t S

ign

al in

ten

sit

y (

MF

I)

82

miR-133a

000

020

040

060

080

100

120


Total RNA Samples

Rela

tive C

han

ge t

o H

eart

FlexmiR v2

qRT-PCR

Tissue

Re

lati

ve

Ch

an

ge


LNP-miRNA

qRT-PCR

83

miRNA

Re

lati

ve

Ch

an

ge



0

5

10

15

20

25


miRNA

Rela

tive C

han

ge

FlexmiR v2

qRT-PCR

LNP-miRNA

qPCR

84



0

5000

10000

15000

20000

25000

30000

hsa-m

iR-1

07

hsa-m

iR-1

47

hsa-m

iR-1

81a

hsa-m

iR-1

92

hsa-m

iR-1

9b

hsa-m

iR-2

20a

hsa-m

iR-2

1hsa-m

iR-2

18

hsa-le

t-7c

hsa-m

iR-1

00

hsa-m

iR-1

25b

hsa-m

iR-

hsa-m

iR-2

05

hsa-m

iR-2

21

hsa-m

iR-2

00a

hsa-m

iR-2

00c

hsa-m

iR-2

06

hsa-m

iR-1

0a

hsa-m

iR-1

6hsa-m

iR-9

hsa-m

iR-4

24

hsa-m

iR-2

0a

hsa-m

iR-2

0b

hsa-le

t-7a

hsa-m

iR-9

5hsa-m

iR-1

36

hsa-m

iR-1

95

hsa-m

iR-2

10

hsa-m

iR-2

12

hsa-m

iR-

hsa-m

iR-1

7hsa-m

iR-1

22

hsa-m

iR-1

43

hsa-m

iR-1

45

hsa-m

ir-1

06a

hsa-m

iR-

hsa-m

iR-1

0b

hsa-m

iR-1

hsa-m

iR-2

23

hsa-m

iR-1

81b

hsa-le

t-155

hsa-le

t-7g

hsa-m

iR-7

ihsa-m

iR-9

6hsa-m

iR-1

37

hsa-m

iR-1

85

hsa-m

iR-1

38

hsa-m

iR-1

33a

hsa-m

iR-1

33b

hsa-m

iR-1

5a

hsa-m

iR-3

4a

hsa-m

iR-2

9a

hsa-m

iR-2

9b

hsa-m

iR-1

03

hsa-m

iR-2

22

hsa-m

iR-1

46a

hsa-m

iR-3

9cel-m

iR-3

9cel-m

iR-2

38

Regions

Ne

t S

ign

al in

ten

sit

y (

MF

I)

50ng

100ng

250ng

500ng








bull Flexible





Proteomic Assays



Nucleic Acids








or IgM





Multiplex Detection

Detection


IgG

1 2 3 4





















Y Y

Y

IgG1 IgG2

IgG3

IgG4 IgA



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Detection



Antigen

1 2 3








Y Y Y

Y

Y

Y Y

Y Y

Y Y

Y



Y


Detection

Multiplex Detection


















compounds





compounds



SAPE SAPE














SAPE SAPE


Detection

Multiplex Detection












543-550






GTPases 827 271-282














Single Plex Assays

To Luminex

Multiplex

Assays









TAG

SAPE

Detection



lt3 Hours Setup











ANNEAL

Ligate

PCR

TAG1

TAG2

cDNA or

gDNAPCR

HYBRIDIZE

amp

LABEL





Upstream LMA Probes

T

Universal Primer 1

5rsquo

A

5rsquo

G



SNP

T



T

T


PCR Amplification

UP 1

P

No Amplification

B-UP 2



Universal Prime mix

UP 1

P

B-UP 2

UP 1

P

B-UP 2

Ligate

T T







TAG

SAPE

Detection



Bead Hyb 80 minutes







Incubation








Targeted PCR




Upstream LDR Probes

A T

G



SNP

A

A



A


Multiple

Ligation

cycles









TAG

SAPE

Detection






Forward primers

gDNA


5rsquo

3rsquo


A

T

5rsquo

3rsquo

G





Targeted PCR



A

5rsquo

3rsquo

G

5rsquo

3rsquo

T




A

5rsquo

3rsquo

G 5rsquo

3rsquo

T








xTAG


Detection

SAPE










xTAG primer

gDNA


Biotinylated primer

5rsquo 3rsquo

3rsquo

dATP dTTP dGTP dCTP

= Carbon Spacer











Detection

Sequence Detection

TAG


















Workflow

75



2 hours



30 minutes



30 minutes





30 minutes

76






0

500

1000

1500

2000

2500

3000

3500

4000


Sample

Sig

nal In

ten

sit

y (

MF

I)

let-7c

Let-7a

77


miR-107 vs miR-103

0

1000

2000

3000

4000

5000

6000


Sample

Sig

na

l In

ten

sit

y (

MF

I)

miR-103

miR-107









0

1000

2000

3000

4000

5000

6000

7000

8000

9000

10000

hsa-m

iR-1

07

hsa-m

iR-1

47

hsa-m

iR-1

81a

hsa-m

iR-1

92

hsa-m

iR-1

9b

hsa-m

iR-2

20a

hsa-m

iR-2

1hsa-m

iR-2

18

hsa-le

t-7c

hsa-m

iR-1

00

hsa-m

iR-1

25b

hsa-m

iR-

hsa-m

iR-2

05

hsa-m

iR-2

21

hsa-m

iR-2

00a

hsa-m

iR-2

00c

hsa-m

iR-2

06

hsa-m

iR-1

0a

hsa-m

iR-1

6

hsa-m

iR-9

hsa-m

iR-4

24

hsa-m

iR-2

0a

hsa-m

iR-2

0b

hsa-le

t-7a

hsa-m

iR-9

5hsa-m

iR-1

36

hsa-m

iR-1

95

hsa-m

iR-2

10

hsa-m

iR-2

12

hsa-m

iR-

hsa-m

iR-1

7

hsa-m

iR-1

22

hsa-m

iR-1

43

hsa-m

iR-1

45

hsa-m

iR-1

06a

hsa-m

iR-

hsa-m

iR-1

0b

hsa-m

iR-1

hsa-m

iR-2

23

hsa-m

iR-1

81b

hsa-m

iR-1

55

hsa-le

t-7g

hsa-le

t-7i

hsa-m

iR-9

6

hsa-m

iR-1

37

hsa-m

iR-1

85

hsa-m

iR-1

38

hsa-m

iR-1

98

hsa-m

iR-1

33a

hsa-m

iR-1

33b

hsa-m

iR-1

5a

hsa-m

iR-3

4a

hsa-m

iR-2

9a

hsa-m

iR-2

9b

hsa-m

iR-1

03

hsa-m

iR-2

22

hsa-m

iR-1

46a

cel-m

iR-3

9cel-m

iR-5

4

cel-m

iR-2

38

Regions

Ne

t S

ign

al In

ten

sit

y (

MF

I)

106a

17


Total RNA Samples

53-Plex Assay

miR-9 miR-122 miR-1

0

5000

10000

15000

20000

25000

Samples

Ne

t S

ign

al In

ten

sit

y (

MF

I)

miR-9

miR-122

miR-1






289704

12431797

4050

7044

12899

18030

24973

23884

21852

19249

11326

6573

3852

2471

0

5000

10000

15000

20000

25000

30000


Input Amount

Ne

t S

ign

al In

ten

sit

y (

MF

I)

200 amol


81


0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000



Ne

t S

ign

al in

ten

sit

y (

MF

I)

82

miR-133a

000

020

040

060

080

100

120


Total RNA Samples

Rela

tive C

han

ge t

o H

eart

FlexmiR v2

qRT-PCR

Tissue

Re

lati

ve

Ch

an

ge


LNP-miRNA

qRT-PCR

83

miRNA

Re

lati

ve

Ch

an

ge



0

5

10

15

20

25


miRNA

Rela

tive C

han

ge

FlexmiR v2

qRT-PCR

LNP-miRNA

qPCR

84



0

5000

10000

15000

20000

25000

30000

hsa-m

iR-1

07

hsa-m

iR-1

47

hsa-m

iR-1

81a

hsa-m

iR-1

92

hsa-m

iR-1

9b

hsa-m

iR-2

20a

hsa-m

iR-2

1hsa-m

iR-2

18

hsa-le

t-7c

hsa-m

iR-1

00

hsa-m

iR-1

25b

hsa-m

iR-

hsa-m

iR-2

05

hsa-m

iR-2

21

hsa-m

iR-2

00a

hsa-m

iR-2

00c

hsa-m

iR-2

06

hsa-m

iR-1

0a

hsa-m

iR-1

6hsa-m

iR-9

hsa-m

iR-4

24

hsa-m

iR-2

0a

hsa-m

iR-2

0b

hsa-le

t-7a

hsa-m

iR-9

5hsa-m

iR-1

36

hsa-m

iR-1

95

hsa-m

iR-2

10

hsa-m

iR-2

12

hsa-m

iR-

hsa-m

iR-1

7hsa-m

iR-1

22

hsa-m

iR-1

43

hsa-m

iR-1

45

hsa-m

ir-1

06a

hsa-m

iR-

hsa-m

iR-1

0b

hsa-m

iR-1

hsa-m

iR-2

23

hsa-m

iR-1

81b

hsa-le

t-155

hsa-le

t-7g

hsa-m

iR-7

ihsa-m

iR-9

6hsa-m

iR-1

37

hsa-m

iR-1

85

hsa-m

iR-1

38

hsa-m

iR-1

33a

hsa-m

iR-1

33b

hsa-m

iR-1

5a

hsa-m

iR-3

4a

hsa-m

iR-2

9a

hsa-m

iR-2

9b

hsa-m

iR-1

03

hsa-m

iR-2

22

hsa-m

iR-1

46a

hsa-m

iR-3

9cel-m

iR-3

9cel-m

iR-2

38

Regions

Ne

t S

ign

al in

ten

sit

y (

MF

I)

50ng

100ng

250ng

500ng








bull Flexible





Proteomic Assays



Nucleic Acids








or IgM





Multiplex Detection

Detection


IgG

1 2 3 4





















Y Y

Y

IgG1 IgG2

IgG3

IgG4 IgA



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Detection



Antigen

1 2 3








Y Y Y

Y

Y

Y Y

Y Y

Y Y

Y



Y


Detection

Multiplex Detection


















compounds





compounds



SAPE SAPE














SAPE SAPE


Detection

Multiplex Detection












543-550






GTPases 827 271-282














Single Plex Assays

To Luminex

Multiplex

Assays





lt3 Hours Setup











ANNEAL

Ligate

PCR

TAG1

TAG2

cDNA or

gDNAPCR

HYBRIDIZE

amp

LABEL





Upstream LMA Probes

T

Universal Primer 1

5rsquo

A

5rsquo

G



SNP

T



T

T


PCR Amplification

UP 1

P

No Amplification

B-UP 2



Universal Prime mix

UP 1

P

B-UP 2

UP 1

P

B-UP 2

Ligate

T T







TAG

SAPE

Detection



Bead Hyb 80 minutes







Incubation








Targeted PCR




Upstream LDR Probes

A T

G



SNP

A

A



A


Multiple

Ligation

cycles









TAG

SAPE

Detection






Forward primers

gDNA


5rsquo

3rsquo


A

T

5rsquo

3rsquo

G





Targeted PCR



A

5rsquo

3rsquo

G

5rsquo

3rsquo

T




A

5rsquo

3rsquo

G 5rsquo

3rsquo

T








xTAG


Detection

SAPE










xTAG primer

gDNA


Biotinylated primer

5rsquo 3rsquo

3rsquo

dATP dTTP dGTP dCTP

= Carbon Spacer











Detection

Sequence Detection

TAG


















Workflow

75



2 hours



30 minutes



30 minutes





30 minutes

76






0

500

1000

1500

2000

2500

3000

3500

4000


Sample

Sig

nal In

ten

sit

y (

MF

I)

let-7c

Let-7a

77


miR-107 vs miR-103

0

1000

2000

3000

4000

5000

6000


Sample

Sig

na

l In

ten

sit

y (

MF

I)

miR-103

miR-107









0

1000

2000

3000

4000

5000

6000

7000

8000

9000

10000

hsa-m

iR-1

07

hsa-m

iR-1

47

hsa-m

iR-1

81a

hsa-m

iR-1

92

hsa-m

iR-1

9b

hsa-m

iR-2

20a

hsa-m

iR-2

1hsa-m

iR-2

18

hsa-le

t-7c

hsa-m

iR-1

00

hsa-m

iR-1

25b

hsa-m

iR-

hsa-m

iR-2

05

hsa-m

iR-2

21

hsa-m

iR-2

00a

hsa-m

iR-2

00c

hsa-m

iR-2

06

hsa-m

iR-1

0a

hsa-m

iR-1

6

hsa-m

iR-9

hsa-m

iR-4

24

hsa-m

iR-2

0a

hsa-m

iR-2

0b

hsa-le

t-7a

hsa-m

iR-9

5hsa-m

iR-1

36

hsa-m

iR-1

95

hsa-m

iR-2

10

hsa-m

iR-2

12

hsa-m

iR-

hsa-m

iR-1

7

hsa-m

iR-1

22

hsa-m

iR-1

43

hsa-m

iR-1

45

hsa-m

iR-1

06a

hsa-m

iR-

hsa-m

iR-1

0b

hsa-m

iR-1

hsa-m

iR-2

23

hsa-m

iR-1

81b

hsa-m

iR-1

55

hsa-le

t-7g

hsa-le

t-7i

hsa-m

iR-9

6

hsa-m

iR-1

37

hsa-m

iR-1

85

hsa-m

iR-1

38

hsa-m

iR-1

98

hsa-m

iR-1

33a

hsa-m

iR-1

33b

hsa-m

iR-1

5a

hsa-m

iR-3

4a

hsa-m

iR-2

9a

hsa-m

iR-2

9b

hsa-m

iR-1

03

hsa-m

iR-2

22

hsa-m

iR-1

46a

cel-m

iR-3

9cel-m

iR-5

4

cel-m

iR-2

38

Regions

Ne

t S

ign

al In

ten

sit

y (

MF

I)

106a

17


Total RNA Samples

53-Plex Assay

miR-9 miR-122 miR-1

0

5000

10000

15000

20000

25000

Samples

Ne

t S

ign

al In

ten

sit

y (

MF

I)

miR-9

miR-122

miR-1






289704

12431797

4050

7044

12899

18030

24973

23884

21852

19249

11326

6573

3852

2471

0

5000

10000

15000

20000

25000

30000


Input Amount

Ne

t S

ign

al In

ten

sit

y (

MF

I)

200 amol


81


0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000



Ne

t S

ign

al in

ten

sit

y (

MF

I)

82

miR-133a

000

020

040

060

080

100

120


Total RNA Samples

Rela

tive C

han

ge t

o H

eart

FlexmiR v2

qRT-PCR

Tissue

Re

lati

ve

Ch

an

ge


LNP-miRNA

qRT-PCR

83

miRNA

Re

lati

ve

Ch

an

ge



0

5

10

15

20

25


miRNA

Rela

tive C

han

ge

FlexmiR v2

qRT-PCR

LNP-miRNA

qPCR

84



0

5000

10000

15000

20000

25000

30000

hsa-m

iR-1

07

hsa-m

iR-1

47

hsa-m

iR-1

81a

hsa-m

iR-1

92

hsa-m

iR-1

9b

hsa-m

iR-2

20a

hsa-m

iR-2

1hsa-m

iR-2

18

hsa-le

t-7c

hsa-m

iR-1

00

hsa-m

iR-1

25b

hsa-m

iR-

hsa-m

iR-2

05

hsa-m

iR-2

21

hsa-m

iR-2

00a

hsa-m

iR-2

00c

hsa-m

iR-2

06

hsa-m

iR-1

0a

hsa-m

iR-1

6hsa-m

iR-9

hsa-m

iR-4

24

hsa-m

iR-2

0a

hsa-m

iR-2

0b

hsa-le

t-7a

hsa-m

iR-9

5hsa-m

iR-1

36

hsa-m

iR-1

95

hsa-m

iR-2

10

hsa-m

iR-2

12

hsa-m

iR-

hsa-m

iR-1

7hsa-m

iR-1

22

hsa-m

iR-1

43

hsa-m

iR-1

45

hsa-m

ir-1

06a

hsa-m

iR-

hsa-m

iR-1

0b

hsa-m

iR-1

hsa-m

iR-2

23

hsa-m

iR-1

81b

hsa-le

t-155

hsa-le

t-7g

hsa-m

iR-7

ihsa-m

iR-9

6hsa-m

iR-1

37

hsa-m

iR-1

85

hsa-m

iR-1

38

hsa-m

iR-1

33a

hsa-m

iR-1

33b

hsa-m

iR-1

5a

hsa-m

iR-3

4a

hsa-m

iR-2

9a

hsa-m

iR-2

9b

hsa-m

iR-1

03

hsa-m

iR-2

22

hsa-m

iR-1

46a

hsa-m

iR-3

9cel-m

iR-3

9cel-m

iR-2

38

Regions

Ne

t S

ign

al in

ten

sit

y (

MF

I)

50ng

100ng

250ng

500ng








bull Flexible





Proteomic Assays



Nucleic Acids








or IgM





Multiplex Detection

Detection


IgG

1 2 3 4





















Y Y

Y

IgG1 IgG2

IgG3

IgG4 IgA



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Detection



Antigen

1 2 3








Y Y Y

Y

Y

Y Y

Y Y

Y Y

Y



Y


Detection

Multiplex Detection


















compounds





compounds



SAPE SAPE














SAPE SAPE


Detection

Multiplex Detection












543-550






GTPases 827 271-282














Single Plex Assays

To Luminex

Multiplex

Assays












ANNEAL

Ligate

PCR

TAG1

TAG2

cDNA or

gDNAPCR

HYBRIDIZE

amp

LABEL





Upstream LMA Probes

T

Universal Primer 1

5rsquo

A

5rsquo

G



SNP

T



T

T


PCR Amplification

UP 1

P

No Amplification

B-UP 2



Universal Prime mix

UP 1

P

B-UP 2

UP 1

P

B-UP 2

Ligate

T T







TAG

SAPE

Detection



Bead Hyb 80 minutes







Incubation








Targeted PCR




Upstream LDR Probes

A T

G



SNP

A

A



A


Multiple

Ligation

cycles









TAG

SAPE

Detection






Forward primers

gDNA


5rsquo

3rsquo


A

T

5rsquo

3rsquo

G





Targeted PCR



A

5rsquo

3rsquo

G

5rsquo

3rsquo

T




A

5rsquo

3rsquo

G 5rsquo

3rsquo

T








xTAG


Detection

SAPE










xTAG primer

gDNA


Biotinylated primer

5rsquo 3rsquo

3rsquo

dATP dTTP dGTP dCTP

= Carbon Spacer











Detection

Sequence Detection

TAG


















Workflow

75



2 hours



30 minutes



30 minutes





30 minutes

76






0

500

1000

1500

2000

2500

3000

3500

4000


Sample

Sig

nal In

ten

sit

y (

MF

I)

let-7c

Let-7a

77


miR-107 vs miR-103

0

1000

2000

3000

4000

5000

6000


Sample

Sig

na

l In

ten

sit

y (

MF

I)

miR-103

miR-107









0

1000

2000

3000

4000

5000

6000

7000

8000

9000

10000

hsa-m

iR-1

07

hsa-m

iR-1

47

hsa-m

iR-1

81a

hsa-m

iR-1

92

hsa-m

iR-1

9b

hsa-m

iR-2

20a

hsa-m

iR-2

1hsa-m

iR-2

18

hsa-le

t-7c

hsa-m

iR-1

00

hsa-m

iR-1

25b

hsa-m

iR-

hsa-m

iR-2

05

hsa-m

iR-2

21

hsa-m

iR-2

00a

hsa-m

iR-2

00c

hsa-m

iR-2

06

hsa-m

iR-1

0a

hsa-m

iR-1

6

hsa-m

iR-9

hsa-m

iR-4

24

hsa-m

iR-2

0a

hsa-m

iR-2

0b

hsa-le

t-7a

hsa-m

iR-9

5hsa-m

iR-1

36

hsa-m

iR-1

95

hsa-m

iR-2

10

hsa-m

iR-2

12

hsa-m

iR-

hsa-m

iR-1

7

hsa-m

iR-1

22

hsa-m

iR-1

43

hsa-m

iR-1

45

hsa-m

iR-1

06a

hsa-m

iR-

hsa-m

iR-1

0b

hsa-m

iR-1

hsa-m

iR-2

23

hsa-m

iR-1

81b

hsa-m

iR-1

55

hsa-le

t-7g

hsa-le

t-7i

hsa-m

iR-9

6

hsa-m

iR-1

37

hsa-m

iR-1

85

hsa-m

iR-1

38

hsa-m

iR-1

98

hsa-m

iR-1

33a

hsa-m

iR-1

33b

hsa-m

iR-1

5a

hsa-m

iR-3

4a

hsa-m

iR-2

9a

hsa-m

iR-2

9b

hsa-m

iR-1

03

hsa-m

iR-2

22

hsa-m

iR-1

46a

cel-m

iR-3

9cel-m

iR-5

4

cel-m

iR-2

38

Regions

Ne

t S

ign

al In

ten

sit

y (

MF

I)

106a

17


Total RNA Samples

53-Plex Assay

miR-9 miR-122 miR-1

0

5000

10000

15000

20000

25000

Samples

Ne

t S

ign

al In

ten

sit

y (

MF

I)

miR-9

miR-122

miR-1






289704

12431797

4050

7044

12899

18030

24973

23884

21852

19249

11326

6573

3852

2471

0

5000

10000

15000

20000

25000

30000


Input Amount

Ne

t S

ign

al In

ten

sit

y (

MF

I)

200 amol


81


0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000



Ne

t S

ign

al in

ten

sit

y (

MF

I)

82

miR-133a

000

020

040

060

080

100

120


Total RNA Samples

Rela

tive C

han

ge t

o H

eart

FlexmiR v2

qRT-PCR

Tissue

Re

lati

ve

Ch

an

ge


LNP-miRNA

qRT-PCR

83

miRNA

Re

lati

ve

Ch

an

ge



0

5

10

15

20

25


miRNA

Rela

tive C

han

ge

FlexmiR v2

qRT-PCR

LNP-miRNA

qPCR

84



0

5000

10000

15000

20000

25000

30000

hsa-m

iR-1

07

hsa-m

iR-1

47

hsa-m

iR-1

81a

hsa-m

iR-1

92

hsa-m

iR-1

9b

hsa-m

iR-2

20a

hsa-m

iR-2

1hsa-m

iR-2

18

hsa-le

t-7c

hsa-m

iR-1

00

hsa-m

iR-1

25b

hsa-m

iR-

hsa-m

iR-2

05

hsa-m

iR-2

21

hsa-m

iR-2

00a

hsa-m

iR-2

00c

hsa-m

iR-2

06

hsa-m

iR-1

0a

hsa-m

iR-1

6hsa-m

iR-9

hsa-m

iR-4

24

hsa-m

iR-2

0a

hsa-m

iR-2

0b

hsa-le

t-7a

hsa-m

iR-9

5hsa-m

iR-1

36

hsa-m

iR-1

95

hsa-m

iR-2

10

hsa-m

iR-2

12

hsa-m

iR-

hsa-m

iR-1

7hsa-m

iR-1

22

hsa-m

iR-1

43

hsa-m

iR-1

45

hsa-m

ir-1

06a

hsa-m

iR-

hsa-m

iR-1

0b

hsa-m

iR-1

hsa-m

iR-2

23

hsa-m

iR-1

81b

hsa-le

t-155

hsa-le

t-7g

hsa-m

iR-7

ihsa-m

iR-9

6hsa-m

iR-1

37

hsa-m

iR-1

85

hsa-m

iR-1

38

hsa-m

iR-1

33a

hsa-m

iR-1

33b

hsa-m

iR-1

5a

hsa-m

iR-3

4a

hsa-m

iR-2

9a

hsa-m

iR-2

9b

hsa-m

iR-1

03

hsa-m

iR-2

22

hsa-m

iR-1

46a

hsa-m

iR-3

9cel-m

iR-3

9cel-m

iR-2

38

Regions

Ne

t S

ign

al in

ten

sit

y (

MF

I)

50ng

100ng

250ng

500ng








bull Flexible





Proteomic Assays



Nucleic Acids








or IgM





Multiplex Detection

Detection


IgG

1 2 3 4





















Y Y

Y

IgG1 IgG2

IgG3

IgG4 IgA



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Detection



Antigen

1 2 3








Y Y Y

Y

Y

Y Y

Y Y

Y Y

Y



Y


Detection

Multiplex Detection


















compounds





compounds



SAPE SAPE














SAPE SAPE


Detection

Multiplex Detection












543-550






GTPases 827 271-282














Single Plex Assays

To Luminex

Multiplex

Assays









ANNEAL

Ligate

PCR

TAG1

TAG2

cDNA or

gDNAPCR

HYBRIDIZE

amp

LABEL





Upstream LMA Probes

T

Universal Primer 1

5rsquo

A

5rsquo

G



SNP

T



T

T


PCR Amplification

UP 1

P

No Amplification

B-UP 2



Universal Prime mix

UP 1

P

B-UP 2

UP 1

P

B-UP 2

Ligate

T T







TAG

SAPE

Detection



Bead Hyb 80 minutes







Incubation








Targeted PCR




Upstream LDR Probes

A T

G



SNP

A

A



A


Multiple

Ligation

cycles









TAG

SAPE

Detection






Forward primers

gDNA


5rsquo

3rsquo


A

T

5rsquo

3rsquo

G





Targeted PCR



A

5rsquo

3rsquo

G

5rsquo

3rsquo

T




A

5rsquo

3rsquo

G 5rsquo

3rsquo

T








xTAG


Detection

SAPE










xTAG primer

gDNA


Biotinylated primer

5rsquo 3rsquo

3rsquo

dATP dTTP dGTP dCTP

= Carbon Spacer











Detection

Sequence Detection

TAG


















Workflow

75



2 hours



30 minutes



30 minutes





30 minutes

76






0

500

1000

1500

2000

2500

3000

3500

4000


Sample

Sig

nal In

ten

sit

y (

MF

I)

let-7c

Let-7a

77


miR-107 vs miR-103

0

1000

2000

3000

4000

5000

6000


Sample

Sig

na

l In

ten

sit

y (

MF

I)

miR-103

miR-107









0

1000

2000

3000

4000

5000

6000

7000

8000

9000

10000

hsa-m

iR-1

07

hsa-m

iR-1

47

hsa-m

iR-1

81a

hsa-m

iR-1

92

hsa-m

iR-1

9b

hsa-m

iR-2

20a

hsa-m

iR-2

1hsa-m

iR-2

18

hsa-le

t-7c

hsa-m

iR-1

00

hsa-m

iR-1

25b

hsa-m

iR-

hsa-m

iR-2

05

hsa-m

iR-2

21

hsa-m

iR-2

00a

hsa-m

iR-2

00c

hsa-m

iR-2

06

hsa-m

iR-1

0a

hsa-m

iR-1

6

hsa-m

iR-9

hsa-m

iR-4

24

hsa-m

iR-2

0a

hsa-m

iR-2

0b

hsa-le

t-7a

hsa-m

iR-9

5hsa-m

iR-1

36

hsa-m

iR-1

95

hsa-m

iR-2

10

hsa-m

iR-2

12

hsa-m

iR-

hsa-m

iR-1

7

hsa-m

iR-1

22

hsa-m

iR-1

43

hsa-m

iR-1

45

hsa-m

iR-1

06a

hsa-m

iR-

hsa-m

iR-1

0b

hsa-m

iR-1

hsa-m

iR-2

23

hsa-m

iR-1

81b

hsa-m

iR-1

55

hsa-le

t-7g

hsa-le

t-7i

hsa-m

iR-9

6

hsa-m

iR-1

37

hsa-m

iR-1

85

hsa-m

iR-1

38

hsa-m

iR-1

98

hsa-m

iR-1

33a

hsa-m

iR-1

33b

hsa-m

iR-1

5a

hsa-m

iR-3

4a

hsa-m

iR-2

9a

hsa-m

iR-2

9b

hsa-m

iR-1

03

hsa-m

iR-2

22

hsa-m

iR-1

46a

cel-m

iR-3

9cel-m

iR-5

4

cel-m

iR-2

38

Regions

Ne

t S

ign

al In

ten

sit

y (

MF

I)

106a

17


Total RNA Samples

53-Plex Assay

miR-9 miR-122 miR-1

0

5000

10000

15000

20000

25000

Samples

Ne

t S

ign

al In

ten

sit

y (

MF

I)

miR-9

miR-122

miR-1






289704

12431797

4050

7044

12899

18030

24973

23884

21852

19249

11326

6573

3852

2471

0

5000

10000

15000

20000

25000

30000


Input Amount

Ne

t S

ign

al In

ten

sit

y (

MF

I)

200 amol


81


0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000



Ne

t S

ign

al in

ten

sit

y (

MF

I)

82

miR-133a

000

020

040

060

080

100

120


Total RNA Samples

Rela

tive C

han

ge t

o H

eart

FlexmiR v2

qRT-PCR

Tissue

Re

lati

ve

Ch

an

ge


LNP-miRNA

qRT-PCR

83

miRNA

Re

lati

ve

Ch

an

ge



0

5

10

15

20

25


miRNA

Rela

tive C

han

ge

FlexmiR v2

qRT-PCR

LNP-miRNA

qPCR

84



0

5000

10000

15000

20000

25000

30000

hsa-m

iR-1

07

hsa-m

iR-1

47

hsa-m

iR-1

81a

hsa-m

iR-1

92

hsa-m

iR-1

9b

hsa-m

iR-2

20a

hsa-m

iR-2

1hsa-m

iR-2

18

hsa-le

t-7c

hsa-m

iR-1

00

hsa-m

iR-1

25b

hsa-m

iR-

hsa-m

iR-2

05

hsa-m

iR-2

21

hsa-m

iR-2

00a

hsa-m

iR-2

00c

hsa-m

iR-2

06

hsa-m

iR-1

0a

hsa-m

iR-1

6hsa-m

iR-9

hsa-m

iR-4

24

hsa-m

iR-2

0a

hsa-m

iR-2

0b

hsa-le

t-7a

hsa-m

iR-9

5hsa-m

iR-1

36

hsa-m

iR-1

95

hsa-m

iR-2

10

hsa-m

iR-2

12

hsa-m

iR-

hsa-m

iR-1

7hsa-m

iR-1

22

hsa-m

iR-1

43

hsa-m

iR-1

45

hsa-m

ir-1

06a

hsa-m

iR-

hsa-m

iR-1

0b

hsa-m

iR-1

hsa-m

iR-2

23

hsa-m

iR-1

81b

hsa-le

t-155

hsa-le

t-7g

hsa-m

iR-7

ihsa-m

iR-9

6hsa-m

iR-1

37

hsa-m

iR-1

85

hsa-m

iR-1

38

hsa-m

iR-1

33a

hsa-m

iR-1

33b

hsa-m

iR-1

5a

hsa-m

iR-3

4a

hsa-m

iR-2

9a

hsa-m

iR-2

9b

hsa-m

iR-1

03

hsa-m

iR-2

22

hsa-m

iR-1

46a

hsa-m

iR-3

9cel-m

iR-3

9cel-m

iR-2

38

Regions

Ne

t S

ign

al in

ten

sit

y (

MF

I)

50ng

100ng

250ng

500ng








bull Flexible





Proteomic Assays



Nucleic Acids








or IgM





Multiplex Detection

Detection


IgG

1 2 3 4





















Y Y

Y

IgG1 IgG2

IgG3

IgG4 IgA



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Detection



Antigen

1 2 3








Y Y Y

Y

Y

Y Y

Y Y

Y Y

Y



Y


Detection

Multiplex Detection


















compounds





compounds



SAPE SAPE














SAPE SAPE


Detection

Multiplex Detection












543-550






GTPases 827 271-282














Single Plex Assays

To Luminex

Multiplex

Assays







ANNEAL

Ligate

PCR

TAG1

TAG2

cDNA or

gDNAPCR

HYBRIDIZE

amp

LABEL





Upstream LMA Probes

T

Universal Primer 1

5rsquo

A

5rsquo

G



SNP

T



T

T


PCR Amplification

UP 1

P

No Amplification

B-UP 2



Universal Prime mix

UP 1

P

B-UP 2

UP 1

P

B-UP 2

Ligate

T T







TAG

SAPE

Detection



Bead Hyb 80 minutes







Incubation








Targeted PCR




Upstream LDR Probes

A T

G



SNP

A

A



A


Multiple

Ligation

cycles









TAG

SAPE

Detection






Forward primers

gDNA


5rsquo

3rsquo


A

T

5rsquo

3rsquo

G





Targeted PCR



A

5rsquo

3rsquo

G

5rsquo

3rsquo

T




A

5rsquo

3rsquo

G 5rsquo

3rsquo

T








xTAG


Detection

SAPE










xTAG primer

gDNA


Biotinylated primer

5rsquo 3rsquo

3rsquo

dATP dTTP dGTP dCTP

= Carbon Spacer











Detection

Sequence Detection

TAG


















Workflow

75



2 hours



30 minutes



30 minutes





30 minutes

76






0

500

1000

1500

2000

2500

3000

3500

4000


Sample

Sig

nal In

ten

sit

y (

MF

I)

let-7c

Let-7a

77


miR-107 vs miR-103

0

1000

2000

3000

4000

5000

6000


Sample

Sig

na

l In

ten

sit

y (

MF

I)

miR-103

miR-107









0

1000

2000

3000

4000

5000

6000

7000

8000

9000

10000

hsa-m

iR-1

07

hsa-m

iR-1

47

hsa-m

iR-1

81a

hsa-m

iR-1

92

hsa-m

iR-1

9b

hsa-m

iR-2

20a

hsa-m

iR-2

1hsa-m

iR-2

18

hsa-le

t-7c

hsa-m

iR-1

00

hsa-m

iR-1

25b

hsa-m

iR-

hsa-m

iR-2

05

hsa-m

iR-2

21

hsa-m

iR-2

00a

hsa-m

iR-2

00c

hsa-m

iR-2

06

hsa-m

iR-1

0a

hsa-m

iR-1

6

hsa-m

iR-9

hsa-m

iR-4

24

hsa-m

iR-2

0a

hsa-m

iR-2

0b

hsa-le

t-7a

hsa-m

iR-9

5hsa-m

iR-1

36

hsa-m

iR-1

95

hsa-m

iR-2

10

hsa-m

iR-2

12

hsa-m

iR-

hsa-m

iR-1

7

hsa-m

iR-1

22

hsa-m

iR-1

43

hsa-m

iR-1

45

hsa-m

iR-1

06a

hsa-m

iR-

hsa-m

iR-1

0b

hsa-m

iR-1

hsa-m

iR-2

23

hsa-m

iR-1

81b

hsa-m

iR-1

55

hsa-le

t-7g

hsa-le

t-7i

hsa-m

iR-9

6

hsa-m

iR-1

37

hsa-m

iR-1

85

hsa-m

iR-1

38

hsa-m

iR-1

98

hsa-m

iR-1

33a

hsa-m

iR-1

33b

hsa-m

iR-1

5a

hsa-m

iR-3

4a

hsa-m

iR-2

9a

hsa-m

iR-2

9b

hsa-m

iR-1

03

hsa-m

iR-2

22

hsa-m

iR-1

46a

cel-m

iR-3

9cel-m

iR-5

4

cel-m

iR-2

38

Regions

Ne

t S

ign

al In

ten

sit

y (

MF

I)

106a

17


Total RNA Samples

53-Plex Assay

miR-9 miR-122 miR-1

0

5000

10000

15000

20000

25000

Samples

Ne

t S

ign

al In

ten

sit

y (

MF

I)

miR-9

miR-122

miR-1






289704

12431797

4050

7044

12899

18030

24973

23884

21852

19249

11326

6573

3852

2471

0

5000

10000

15000

20000

25000

30000


Input Amount

Ne

t S

ign

al In

ten

sit

y (

MF

I)

200 amol


81


0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000



Ne

t S

ign

al in

ten

sit

y (

MF

I)

82

miR-133a

000

020

040

060

080

100

120


Total RNA Samples

Rela

tive C

han

ge t

o H

eart

FlexmiR v2

qRT-PCR

Tissue

Re

lati

ve

Ch

an

ge


LNP-miRNA

qRT-PCR

83

miRNA

Re

lati

ve

Ch

an

ge



0

5

10

15

20

25


miRNA

Rela

tive C

han

ge

FlexmiR v2

qRT-PCR

LNP-miRNA

qPCR

84



0

5000

10000

15000

20000

25000

30000

hsa-m

iR-1

07

hsa-m

iR-1

47

hsa-m

iR-1

81a

hsa-m

iR-1

92

hsa-m

iR-1

9b

hsa-m

iR-2

20a

hsa-m

iR-2

1hsa-m

iR-2

18

hsa-le

t-7c

hsa-m

iR-1

00

hsa-m

iR-1

25b

hsa-m

iR-

hsa-m

iR-2

05

hsa-m

iR-2

21

hsa-m

iR-2

00a

hsa-m

iR-2

00c

hsa-m

iR-2

06

hsa-m

iR-1

0a

hsa-m

iR-1

6hsa-m

iR-9

hsa-m

iR-4

24

hsa-m

iR-2

0a

hsa-m

iR-2

0b

hsa-le

t-7a

hsa-m

iR-9

5hsa-m

iR-1

36

hsa-m

iR-1

95

hsa-m

iR-2

10

hsa-m

iR-2

12

hsa-m

iR-

hsa-m

iR-1

7hsa-m

iR-1

22

hsa-m

iR-1

43

hsa-m

iR-1

45

hsa-m

ir-1

06a

hsa-m

iR-

hsa-m

iR-1

0b

hsa-m

iR-1

hsa-m

iR-2

23

hsa-m

iR-1

81b

hsa-le

t-155

hsa-le

t-7g

hsa-m

iR-7

ihsa-m

iR-9

6hsa-m

iR-1

37

hsa-m

iR-1

85

hsa-m

iR-1

38

hsa-m

iR-1

33a

hsa-m

iR-1

33b

hsa-m

iR-1

5a

hsa-m

iR-3

4a

hsa-m

iR-2

9a

hsa-m

iR-2

9b

hsa-m

iR-1

03

hsa-m

iR-2

22

hsa-m

iR-1

46a

hsa-m

iR-3

9cel-m

iR-3

9cel-m

iR-2

38

Regions

Ne

t S

ign

al in

ten

sit

y (

MF

I)

50ng

100ng

250ng

500ng








bull Flexible





Proteomic Assays



Nucleic Acids








or IgM





Multiplex Detection

Detection


IgG

1 2 3 4





















Y Y

Y

IgG1 IgG2

IgG3

IgG4 IgA



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Detection



Antigen

1 2 3








Y Y Y

Y

Y

Y Y

Y Y

Y Y

Y



Y


Detection

Multiplex Detection


















compounds





compounds



SAPE SAPE














SAPE SAPE


Detection

Multiplex Detection












543-550






GTPases 827 271-282














Single Plex Assays

To Luminex

Multiplex

Assays






ANNEAL

Ligate

PCR

TAG1

TAG2

cDNA or

gDNAPCR

HYBRIDIZE

amp

LABEL





Upstream LMA Probes

T

Universal Primer 1

5rsquo

A

5rsquo

G



SNP

T



T

T


PCR Amplification

UP 1

P

No Amplification

B-UP 2



Universal Prime mix

UP 1

P

B-UP 2

UP 1

P

B-UP 2

Ligate

T T







TAG

SAPE

Detection



Bead Hyb 80 minutes







Incubation








Targeted PCR




Upstream LDR Probes

A T

G



SNP

A

A



A


Multiple

Ligation

cycles









TAG

SAPE

Detection






Forward primers

gDNA


5rsquo

3rsquo


A

T

5rsquo

3rsquo

G





Targeted PCR



A

5rsquo

3rsquo

G

5rsquo

3rsquo

T




A

5rsquo

3rsquo

G 5rsquo

3rsquo

T








xTAG


Detection

SAPE










xTAG primer

gDNA


Biotinylated primer

5rsquo 3rsquo

3rsquo

dATP dTTP dGTP dCTP

= Carbon Spacer











Detection

Sequence Detection

TAG


















Workflow

75



2 hours



30 minutes



30 minutes





30 minutes

76






0

500

1000

1500

2000

2500

3000

3500

4000


Sample

Sig

nal In

ten

sit

y (

MF

I)

let-7c

Let-7a

77


miR-107 vs miR-103

0

1000

2000

3000

4000

5000

6000


Sample

Sig

na

l In

ten

sit

y (

MF

I)

miR-103

miR-107









0

1000

2000

3000

4000

5000

6000

7000

8000

9000

10000

hsa-m

iR-1

07

hsa-m

iR-1

47

hsa-m

iR-1

81a

hsa-m

iR-1

92

hsa-m

iR-1

9b

hsa-m

iR-2

20a

hsa-m

iR-2

1hsa-m

iR-2

18

hsa-le

t-7c

hsa-m

iR-1

00

hsa-m

iR-1

25b

hsa-m

iR-

hsa-m

iR-2

05

hsa-m

iR-2

21

hsa-m

iR-2

00a

hsa-m

iR-2

00c

hsa-m

iR-2

06

hsa-m

iR-1

0a

hsa-m

iR-1

6

hsa-m

iR-9

hsa-m

iR-4

24

hsa-m

iR-2

0a

hsa-m

iR-2

0b

hsa-le

t-7a

hsa-m

iR-9

5hsa-m

iR-1

36

hsa-m

iR-1

95

hsa-m

iR-2

10

hsa-m

iR-2

12

hsa-m

iR-

hsa-m

iR-1

7

hsa-m

iR-1

22

hsa-m

iR-1

43

hsa-m

iR-1

45

hsa-m

iR-1

06a

hsa-m

iR-

hsa-m

iR-1

0b

hsa-m

iR-1

hsa-m

iR-2

23

hsa-m

iR-1

81b

hsa-m

iR-1

55

hsa-le

t-7g

hsa-le

t-7i

hsa-m

iR-9

6

hsa-m

iR-1

37

hsa-m

iR-1

85

hsa-m

iR-1

38

hsa-m

iR-1

98

hsa-m

iR-1

33a

hsa-m

iR-1

33b

hsa-m

iR-1

5a

hsa-m

iR-3

4a

hsa-m

iR-2

9a

hsa-m

iR-2

9b

hsa-m

iR-1

03

hsa-m

iR-2

22

hsa-m

iR-1

46a

cel-m

iR-3

9cel-m

iR-5

4

cel-m

iR-2

38

Regions

Ne

t S

ign

al In

ten

sit

y (

MF

I)

106a

17


Total RNA Samples

53-Plex Assay

miR-9 miR-122 miR-1

0

5000

10000

15000

20000

25000

Samples

Ne

t S

ign

al In

ten

sit

y (

MF

I)

miR-9

miR-122

miR-1






289704

12431797

4050

7044

12899

18030

24973

23884

21852

19249

11326

6573

3852

2471

0

5000

10000

15000

20000

25000

30000


Input Amount

Ne

t S

ign

al In

ten

sit

y (

MF

I)

200 amol


81


0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000



Ne

t S

ign

al in

ten

sit

y (

MF

I)

82

miR-133a

000

020

040

060

080

100

120


Total RNA Samples

Rela

tive C

han

ge t

o H

eart

FlexmiR v2

qRT-PCR

Tissue

Re

lati

ve

Ch

an

ge


LNP-miRNA

qRT-PCR

83

miRNA

Re

lati

ve

Ch

an

ge



0

5

10

15

20

25


miRNA

Rela

tive C

han

ge

FlexmiR v2

qRT-PCR

LNP-miRNA

qPCR

84



0

5000

10000

15000

20000

25000

30000

hsa-m

iR-1

07

hsa-m

iR-1

47

hsa-m

iR-1

81a

hsa-m

iR-1

92

hsa-m

iR-1

9b

hsa-m

iR-2

20a

hsa-m

iR-2

1hsa-m

iR-2

18

hsa-le

t-7c

hsa-m

iR-1

00

hsa-m

iR-1

25b

hsa-m

iR-

hsa-m

iR-2

05

hsa-m

iR-2

21

hsa-m

iR-2

00a

hsa-m

iR-2

00c

hsa-m

iR-2

06

hsa-m

iR-1

0a

hsa-m

iR-1

6hsa-m

iR-9

hsa-m

iR-4

24

hsa-m

iR-2

0a

hsa-m

iR-2

0b

hsa-le

t-7a

hsa-m

iR-9

5hsa-m

iR-1

36

hsa-m

iR-1

95

hsa-m

iR-2

10

hsa-m

iR-2

12

hsa-m

iR-

hsa-m

iR-1

7hsa-m

iR-1

22

hsa-m

iR-1

43

hsa-m

iR-1

45

hsa-m

ir-1

06a

hsa-m

iR-

hsa-m

iR-1

0b

hsa-m

iR-1

hsa-m

iR-2

23

hsa-m

iR-1

81b

hsa-le

t-155

hsa-le

t-7g

hsa-m

iR-7

ihsa-m

iR-9

6hsa-m

iR-1

37

hsa-m

iR-1

85

hsa-m

iR-1

38

hsa-m

iR-1

33a

hsa-m

iR-1

33b

hsa-m

iR-1

5a

hsa-m

iR-3

4a

hsa-m

iR-2

9a

hsa-m

iR-2

9b

hsa-m

iR-1

03

hsa-m

iR-2

22

hsa-m

iR-1

46a

hsa-m

iR-3

9cel-m

iR-3

9cel-m

iR-2

38

Regions

Ne

t S

ign

al in

ten

sit

y (

MF

I)

50ng

100ng

250ng

500ng








bull Flexible





Proteomic Assays



Nucleic Acids








or IgM





Multiplex Detection

Detection


IgG

1 2 3 4





















Y Y

Y

IgG1 IgG2

IgG3

IgG4 IgA



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Detection



Antigen

1 2 3








Y Y Y

Y

Y

Y Y

Y Y

Y Y

Y



Y


Detection

Multiplex Detection


















compounds





compounds



SAPE SAPE














SAPE SAPE


Detection

Multiplex Detection












543-550






GTPases 827 271-282














Single Plex Assays

To Luminex

Multiplex

Assays







Upstream LMA Probes

T

Universal Primer 1

5rsquo

A

5rsquo

G



SNP

T



T

T


PCR Amplification

UP 1

P

No Amplification

B-UP 2



Universal Prime mix

UP 1

P

B-UP 2

UP 1

P

B-UP 2

Ligate

T T







TAG

SAPE

Detection



Bead Hyb 80 minutes







Incubation








Targeted PCR




Upstream LDR Probes

A T

G



SNP

A

A



A


Multiple

Ligation

cycles









TAG

SAPE

Detection






Forward primers

gDNA


5rsquo

3rsquo


A

T

5rsquo

3rsquo

G





Targeted PCR



A

5rsquo

3rsquo

G

5rsquo

3rsquo

T




A

5rsquo

3rsquo

G 5rsquo

3rsquo

T








xTAG


Detection

SAPE










xTAG primer

gDNA


Biotinylated primer

5rsquo 3rsquo

3rsquo

dATP dTTP dGTP dCTP

= Carbon Spacer











Detection

Sequence Detection

TAG


















Workflow

75



2 hours



30 minutes



30 minutes





30 minutes

76






0

500

1000

1500

2000

2500

3000

3500

4000


Sample

Sig

nal In

ten

sit

y (

MF

I)

let-7c

Let-7a

77


miR-107 vs miR-103

0

1000

2000

3000

4000

5000

6000


Sample

Sig

na

l In

ten

sit

y (

MF

I)

miR-103

miR-107









0

1000

2000

3000

4000

5000

6000

7000

8000

9000

10000

hsa-m

iR-1

07

hsa-m

iR-1

47

hsa-m

iR-1

81a

hsa-m

iR-1

92

hsa-m

iR-1

9b

hsa-m

iR-2

20a

hsa-m

iR-2

1hsa-m

iR-2

18

hsa-le

t-7c

hsa-m

iR-1

00

hsa-m

iR-1

25b

hsa-m

iR-

hsa-m

iR-2

05

hsa-m

iR-2

21

hsa-m

iR-2

00a

hsa-m

iR-2

00c

hsa-m

iR-2

06

hsa-m

iR-1

0a

hsa-m

iR-1

6

hsa-m

iR-9

hsa-m

iR-4

24

hsa-m

iR-2

0a

hsa-m

iR-2

0b

hsa-le

t-7a

hsa-m

iR-9

5hsa-m

iR-1

36

hsa-m

iR-1

95

hsa-m

iR-2

10

hsa-m

iR-2

12

hsa-m

iR-

hsa-m

iR-1

7

hsa-m

iR-1

22

hsa-m

iR-1

43

hsa-m

iR-1

45

hsa-m

iR-1

06a

hsa-m

iR-

hsa-m

iR-1

0b

hsa-m

iR-1

hsa-m

iR-2

23

hsa-m

iR-1

81b

hsa-m

iR-1

55

hsa-le

t-7g

hsa-le

t-7i

hsa-m

iR-9

6

hsa-m

iR-1

37

hsa-m

iR-1

85

hsa-m

iR-1

38

hsa-m

iR-1

98

hsa-m

iR-1

33a

hsa-m

iR-1

33b

hsa-m

iR-1

5a

hsa-m

iR-3

4a

hsa-m

iR-2

9a

hsa-m

iR-2

9b

hsa-m

iR-1

03

hsa-m

iR-2

22

hsa-m

iR-1

46a

cel-m

iR-3

9cel-m

iR-5

4

cel-m

iR-2

38

Regions

Ne

t S

ign

al In

ten

sit

y (

MF

I)

106a

17


Total RNA Samples

53-Plex Assay

miR-9 miR-122 miR-1

0

5000

10000

15000

20000

25000

Samples

Ne

t S

ign

al In

ten

sit

y (

MF

I)

miR-9

miR-122

miR-1






289704

12431797

4050

7044

12899

18030

24973

23884

21852

19249

11326

6573

3852

2471

0

5000

10000

15000

20000

25000

30000


Input Amount

Ne

t S

ign

al In

ten

sit

y (

MF

I)

200 amol


81


0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000



Ne

t S

ign

al in

ten

sit

y (

MF

I)

82

miR-133a

000

020

040

060

080

100

120


Total RNA Samples

Rela

tive C

han

ge t

o H

eart

FlexmiR v2

qRT-PCR

Tissue

Re

lati

ve

Ch

an

ge


LNP-miRNA

qRT-PCR

83

miRNA

Re

lati

ve

Ch

an

ge



0

5

10

15

20

25


miRNA

Rela

tive C

han

ge

FlexmiR v2

qRT-PCR

LNP-miRNA

qPCR

84



0

5000

10000

15000

20000

25000

30000

hsa-m

iR-1

07

hsa-m

iR-1

47

hsa-m

iR-1

81a

hsa-m

iR-1

92

hsa-m

iR-1

9b

hsa-m

iR-2

20a

hsa-m

iR-2

1hsa-m

iR-2

18

hsa-le

t-7c

hsa-m

iR-1

00

hsa-m

iR-1

25b

hsa-m

iR-

hsa-m

iR-2

05

hsa-m

iR-2

21

hsa-m

iR-2

00a

hsa-m

iR-2

00c

hsa-m

iR-2

06

hsa-m

iR-1

0a

hsa-m

iR-1

6hsa-m

iR-9

hsa-m

iR-4

24

hsa-m

iR-2

0a

hsa-m

iR-2

0b

hsa-le

t-7a

hsa-m

iR-9

5hsa-m

iR-1

36

hsa-m

iR-1

95

hsa-m

iR-2

10

hsa-m

iR-2

12

hsa-m

iR-

hsa-m

iR-1

7hsa-m

iR-1

22

hsa-m

iR-1

43

hsa-m

iR-1

45

hsa-m

ir-1

06a

hsa-m

iR-

hsa-m

iR-1

0b

hsa-m

iR-1

hsa-m

iR-2

23

hsa-m

iR-1

81b

hsa-le

t-155

hsa-le

t-7g

hsa-m

iR-7

ihsa-m

iR-9

6hsa-m

iR-1

37

hsa-m

iR-1

85

hsa-m

iR-1

38

hsa-m

iR-1

33a

hsa-m

iR-1

33b

hsa-m

iR-1

5a

hsa-m

iR-3

4a

hsa-m

iR-2

9a

hsa-m

iR-2

9b

hsa-m

iR-1

03

hsa-m

iR-2

22

hsa-m

iR-1

46a

hsa-m

iR-3

9cel-m

iR-3

9cel-m

iR-2

38

Regions

Ne

t S

ign

al in

ten

sit

y (

MF

I)

50ng

100ng

250ng

500ng








bull Flexible





Proteomic Assays



Nucleic Acids








or IgM





Multiplex Detection

Detection


IgG

1 2 3 4





















Y Y

Y

IgG1 IgG2

IgG3

IgG4 IgA



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Detection



Antigen

1 2 3








Y Y Y

Y

Y

Y Y

Y Y

Y Y

Y



Y


Detection

Multiplex Detection


















compounds





compounds



SAPE SAPE














SAPE SAPE


Detection

Multiplex Detection












543-550






GTPases 827 271-282














Single Plex Assays

To Luminex

Multiplex

Assays






Upstream LMA Probes

T

Universal Primer 1

5rsquo

A

5rsquo

G



SNP

T



T

T


PCR Amplification

UP 1

P

No Amplification

B-UP 2



Universal Prime mix

UP 1

P

B-UP 2

UP 1

P

B-UP 2

Ligate

T T







TAG

SAPE

Detection



Bead Hyb 80 minutes







Incubation








Targeted PCR




Upstream LDR Probes

A T

G



SNP

A

A



A


Multiple

Ligation

cycles









TAG

SAPE

Detection






Forward primers

gDNA


5rsquo

3rsquo


A

T

5rsquo

3rsquo

G





Targeted PCR



A

5rsquo

3rsquo

G

5rsquo

3rsquo

T




A

5rsquo

3rsquo

G 5rsquo

3rsquo

T








xTAG


Detection

SAPE










xTAG primer

gDNA


Biotinylated primer

5rsquo 3rsquo

3rsquo

dATP dTTP dGTP dCTP

= Carbon Spacer











Detection

Sequence Detection

TAG


















Workflow

75



2 hours



30 minutes



30 minutes





30 minutes

76






0

500

1000

1500

2000

2500

3000

3500

4000


Sample

Sig

nal In

ten

sit

y (

MF

I)

let-7c

Let-7a

77


miR-107 vs miR-103

0

1000

2000

3000

4000

5000

6000


Sample

Sig

na

l In

ten

sit

y (

MF

I)

miR-103

miR-107









0

1000

2000

3000

4000

5000

6000

7000

8000

9000

10000

hsa-m

iR-1

07

hsa-m

iR-1

47

hsa-m

iR-1

81a

hsa-m

iR-1

92

hsa-m

iR-1

9b

hsa-m

iR-2

20a

hsa-m

iR-2

1hsa-m

iR-2

18

hsa-le

t-7c

hsa-m

iR-1

00

hsa-m

iR-1

25b

hsa-m

iR-

hsa-m

iR-2

05

hsa-m

iR-2

21

hsa-m

iR-2

00a

hsa-m

iR-2

00c

hsa-m

iR-2

06

hsa-m

iR-1

0a

hsa-m

iR-1

6

hsa-m

iR-9

hsa-m

iR-4

24

hsa-m

iR-2

0a

hsa-m

iR-2

0b

hsa-le

t-7a

hsa-m

iR-9

5hsa-m

iR-1

36

hsa-m

iR-1

95

hsa-m

iR-2

10

hsa-m

iR-2

12

hsa-m

iR-

hsa-m

iR-1

7

hsa-m

iR-1

22

hsa-m

iR-1

43

hsa-m

iR-1

45

hsa-m

iR-1

06a

hsa-m

iR-

hsa-m

iR-1

0b

hsa-m

iR-1

hsa-m

iR-2

23

hsa-m

iR-1

81b

hsa-m

iR-1

55

hsa-le

t-7g

hsa-le

t-7i

hsa-m

iR-9

6

hsa-m

iR-1

37

hsa-m

iR-1

85

hsa-m

iR-1

38

hsa-m

iR-1

98

hsa-m

iR-1

33a

hsa-m

iR-1

33b

hsa-m

iR-1

5a

hsa-m

iR-3

4a

hsa-m

iR-2

9a

hsa-m

iR-2

9b

hsa-m

iR-1

03

hsa-m

iR-2

22

hsa-m

iR-1

46a

cel-m

iR-3

9cel-m

iR-5

4

cel-m

iR-2

38

Regions

Ne

t S

ign

al In

ten

sit

y (

MF

I)

106a

17


Total RNA Samples

53-Plex Assay

miR-9 miR-122 miR-1

0

5000

10000

15000

20000

25000

Samples

Ne

t S

ign

al In

ten

sit

y (

MF

I)

miR-9

miR-122

miR-1






289704

12431797

4050

7044

12899

18030

24973

23884

21852

19249

11326

6573

3852

2471

0

5000

10000

15000

20000

25000

30000


Input Amount

Ne

t S

ign

al In

ten

sit

y (

MF

I)

200 amol


81


0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000



Ne

t S

ign

al in

ten

sit

y (

MF

I)

82

miR-133a

000

020

040

060

080

100

120


Total RNA Samples

Rela

tive C

han

ge t

o H

eart

FlexmiR v2

qRT-PCR

Tissue

Re

lati

ve

Ch

an

ge


LNP-miRNA

qRT-PCR

83

miRNA

Re

lati

ve

Ch

an

ge



0

5

10

15

20

25


miRNA

Rela

tive C

han

ge

FlexmiR v2

qRT-PCR

LNP-miRNA

qPCR

84



0

5000

10000

15000

20000

25000

30000

hsa-m

iR-1

07

hsa-m

iR-1

47

hsa-m

iR-1

81a

hsa-m

iR-1

92

hsa-m

iR-1

9b

hsa-m

iR-2

20a

hsa-m

iR-2

1hsa-m

iR-2

18

hsa-le

t-7c

hsa-m

iR-1

00

hsa-m

iR-1

25b

hsa-m

iR-

hsa-m

iR-2

05

hsa-m

iR-2

21

hsa-m

iR-2

00a

hsa-m

iR-2

00c

hsa-m

iR-2

06

hsa-m

iR-1

0a

hsa-m

iR-1

6hsa-m

iR-9

hsa-m

iR-4

24

hsa-m

iR-2

0a

hsa-m

iR-2

0b

hsa-le

t-7a

hsa-m

iR-9

5hsa-m

iR-1

36

hsa-m

iR-1

95

hsa-m

iR-2

10

hsa-m

iR-2

12

hsa-m

iR-

hsa-m

iR-1

7hsa-m

iR-1

22

hsa-m

iR-1

43

hsa-m

iR-1

45

hsa-m

ir-1

06a

hsa-m

iR-

hsa-m

iR-1

0b

hsa-m

iR-1

hsa-m

iR-2

23

hsa-m

iR-1

81b

hsa-le

t-155

hsa-le

t-7g

hsa-m

iR-7

ihsa-m

iR-9

6hsa-m

iR-1

37

hsa-m

iR-1

85

hsa-m

iR-1

38

hsa-m

iR-1

33a

hsa-m

iR-1

33b

hsa-m

iR-1

5a

hsa-m

iR-3

4a

hsa-m

iR-2

9a

hsa-m

iR-2

9b

hsa-m

iR-1

03

hsa-m

iR-2

22

hsa-m

iR-1

46a

hsa-m

iR-3

9cel-m

iR-3

9cel-m

iR-2

38

Regions

Ne

t S

ign

al in

ten

sit

y (

MF

I)

50ng

100ng

250ng

500ng








bull Flexible





Proteomic Assays



Nucleic Acids








or IgM





Multiplex Detection

Detection


IgG

1 2 3 4





















Y Y

Y

IgG1 IgG2

IgG3

IgG4 IgA



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Detection



Antigen

1 2 3








Y Y Y

Y

Y

Y Y

Y Y

Y Y

Y



Y


Detection

Multiplex Detection


















compounds





compounds



SAPE SAPE














SAPE SAPE


Detection

Multiplex Detection












543-550






GTPases 827 271-282














Single Plex Assays

To Luminex

Multiplex

Assays





SNP

T



T

T


PCR Amplification

UP 1

P

No Amplification

B-UP 2



Universal Prime mix

UP 1

P

B-UP 2

UP 1

P

B-UP 2

Ligate

T T







TAG

SAPE

Detection



Bead Hyb 80 minutes







Incubation








Targeted PCR




Upstream LDR Probes

A T

G



SNP

A

A



A


Multiple

Ligation

cycles









TAG

SAPE

Detection






Forward primers

gDNA


5rsquo

3rsquo


A

T

5rsquo

3rsquo

G





Targeted PCR



A

5rsquo

3rsquo

G

5rsquo

3rsquo

T




A

5rsquo

3rsquo

G 5rsquo

3rsquo

T








xTAG


Detection

SAPE










xTAG primer

gDNA


Biotinylated primer

5rsquo 3rsquo

3rsquo

dATP dTTP dGTP dCTP

= Carbon Spacer











Detection

Sequence Detection

TAG


















Workflow

75



2 hours



30 minutes



30 minutes





30 minutes

76






0

500

1000

1500

2000

2500

3000

3500

4000


Sample

Sig

nal In

ten

sit

y (

MF

I)

let-7c

Let-7a

77


miR-107 vs miR-103

0

1000

2000

3000

4000

5000

6000


Sample

Sig

na

l In

ten

sit

y (

MF

I)

miR-103

miR-107









0

1000

2000

3000

4000

5000

6000

7000

8000

9000

10000

hsa-m

iR-1

07

hsa-m

iR-1

47

hsa-m

iR-1

81a

hsa-m

iR-1

92

hsa-m

iR-1

9b

hsa-m

iR-2

20a

hsa-m

iR-2

1hsa-m

iR-2

18

hsa-le

t-7c

hsa-m

iR-1

00

hsa-m

iR-1

25b

hsa-m

iR-

hsa-m

iR-2

05

hsa-m

iR-2

21

hsa-m

iR-2

00a

hsa-m

iR-2

00c

hsa-m

iR-2

06

hsa-m

iR-1

0a

hsa-m

iR-1

6

hsa-m

iR-9

hsa-m

iR-4

24

hsa-m

iR-2

0a

hsa-m

iR-2

0b

hsa-le

t-7a

hsa-m

iR-9

5hsa-m

iR-1

36

hsa-m

iR-1

95

hsa-m

iR-2

10

hsa-m

iR-2

12

hsa-m

iR-

hsa-m

iR-1

7

hsa-m

iR-1

22

hsa-m

iR-1

43

hsa-m

iR-1

45

hsa-m

iR-1

06a

hsa-m

iR-

hsa-m

iR-1

0b

hsa-m

iR-1

hsa-m

iR-2

23

hsa-m

iR-1

81b

hsa-m

iR-1

55

hsa-le

t-7g

hsa-le

t-7i

hsa-m

iR-9

6

hsa-m

iR-1

37

hsa-m

iR-1

85

hsa-m

iR-1

38

hsa-m

iR-1

98

hsa-m

iR-1

33a

hsa-m

iR-1

33b

hsa-m

iR-1

5a

hsa-m

iR-3

4a

hsa-m

iR-2

9a

hsa-m

iR-2

9b

hsa-m

iR-1

03

hsa-m

iR-2

22

hsa-m

iR-1

46a

cel-m

iR-3

9cel-m

iR-5

4

cel-m

iR-2

38

Regions

Ne

t S

ign

al In

ten

sit

y (

MF

I)

106a

17


Total RNA Samples

53-Plex Assay

miR-9 miR-122 miR-1

0

5000

10000

15000

20000

25000

Samples

Ne

t S

ign

al In

ten

sit

y (

MF

I)

miR-9

miR-122

miR-1






289704

12431797

4050

7044

12899

18030

24973

23884

21852

19249

11326

6573

3852

2471

0

5000

10000

15000

20000

25000

30000


Input Amount

Ne

t S

ign

al In

ten

sit

y (

MF

I)

200 amol


81


0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000



Ne

t S

ign

al in

ten

sit

y (

MF

I)

82

miR-133a

000

020

040

060

080

100

120


Total RNA Samples

Rela

tive C

han

ge t

o H

eart

FlexmiR v2

qRT-PCR

Tissue

Re

lati

ve

Ch

an

ge


LNP-miRNA

qRT-PCR

83

miRNA

Re

lati

ve

Ch

an

ge



0

5

10

15

20

25


miRNA

Rela

tive C

han

ge

FlexmiR v2

qRT-PCR

LNP-miRNA

qPCR

84



0

5000

10000

15000

20000

25000

30000

hsa-m

iR-1

07

hsa-m

iR-1

47

hsa-m

iR-1

81a

hsa-m

iR-1

92

hsa-m

iR-1

9b

hsa-m

iR-2

20a

hsa-m

iR-2

1hsa-m

iR-2

18

hsa-le

t-7c

hsa-m

iR-1

00

hsa-m

iR-1

25b

hsa-m

iR-

hsa-m

iR-2

05

hsa-m

iR-2

21

hsa-m

iR-2

00a

hsa-m

iR-2

00c

hsa-m

iR-2

06

hsa-m

iR-1

0a

hsa-m

iR-1

6hsa-m

iR-9

hsa-m

iR-4

24

hsa-m

iR-2

0a

hsa-m

iR-2

0b

hsa-le

t-7a

hsa-m

iR-9

5hsa-m

iR-1

36

hsa-m

iR-1

95

hsa-m

iR-2

10

hsa-m

iR-2

12

hsa-m

iR-

hsa-m

iR-1

7hsa-m

iR-1

22

hsa-m

iR-1

43

hsa-m

iR-1

45

hsa-m

ir-1

06a

hsa-m

iR-

hsa-m

iR-1

0b

hsa-m

iR-1

hsa-m

iR-2

23

hsa-m

iR-1

81b

hsa-le

t-155

hsa-le

t-7g

hsa-m

iR-7

ihsa-m

iR-9

6hsa-m

iR-1

37

hsa-m

iR-1

85

hsa-m

iR-1

38

hsa-m

iR-1

33a

hsa-m

iR-1

33b

hsa-m

iR-1

5a

hsa-m

iR-3

4a

hsa-m

iR-2

9a

hsa-m

iR-2

9b

hsa-m

iR-1

03

hsa-m

iR-2

22

hsa-m

iR-1

46a

hsa-m

iR-3

9cel-m

iR-3

9cel-m

iR-2

38

Regions

Ne

t S

ign

al in

ten

sit

y (

MF

I)

50ng

100ng

250ng

500ng








bull Flexible





Proteomic Assays



Nucleic Acids








or IgM





Multiplex Detection

Detection


IgG

1 2 3 4





















Y Y

Y

IgG1 IgG2

IgG3

IgG4 IgA



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Detection



Antigen

1 2 3








Y Y Y

Y

Y

Y Y

Y Y

Y Y

Y



Y


Detection

Multiplex Detection


















compounds





compounds



SAPE SAPE














SAPE SAPE


Detection

Multiplex Detection












543-550






GTPases 827 271-282














Single Plex Assays

To Luminex

Multiplex

Assays




PCR Amplification

UP 1

P

No Amplification

B-UP 2



Universal Prime mix

UP 1

P

B-UP 2

UP 1

P

B-UP 2

Ligate

T T







TAG

SAPE

Detection



Bead Hyb 80 minutes







Incubation








Targeted PCR




Upstream LDR Probes

A T

G



SNP

A

A



A


Multiple

Ligation

cycles









TAG

SAPE

Detection






Forward primers

gDNA


5rsquo

3rsquo


A

T

5rsquo

3rsquo

G





Targeted PCR



A

5rsquo

3rsquo

G

5rsquo

3rsquo

T




A

5rsquo

3rsquo

G 5rsquo

3rsquo

T








xTAG


Detection

SAPE










xTAG primer

gDNA


Biotinylated primer

5rsquo 3rsquo

3rsquo

dATP dTTP dGTP dCTP

= Carbon Spacer











Detection

Sequence Detection

TAG


















Workflow

75



2 hours



30 minutes



30 minutes





30 minutes

76






0

500

1000

1500

2000

2500

3000

3500

4000


Sample

Sig

nal In

ten

sit

y (

MF

I)

let-7c

Let-7a

77


miR-107 vs miR-103

0

1000

2000

3000

4000

5000

6000


Sample

Sig

na

l In

ten

sit

y (

MF

I)

miR-103

miR-107









0

1000

2000

3000

4000

5000

6000

7000

8000

9000

10000

hsa-m

iR-1

07

hsa-m

iR-1

47

hsa-m

iR-1

81a

hsa-m

iR-1

92

hsa-m

iR-1

9b

hsa-m

iR-2

20a

hsa-m

iR-2

1hsa-m

iR-2

18

hsa-le

t-7c

hsa-m

iR-1

00

hsa-m

iR-1

25b

hsa-m

iR-

hsa-m

iR-2

05

hsa-m

iR-2

21

hsa-m

iR-2

00a

hsa-m

iR-2

00c

hsa-m

iR-2

06

hsa-m

iR-1

0a

hsa-m

iR-1

6

hsa-m

iR-9

hsa-m

iR-4

24

hsa-m

iR-2

0a

hsa-m

iR-2

0b

hsa-le

t-7a

hsa-m

iR-9

5hsa-m

iR-1

36

hsa-m

iR-1

95

hsa-m

iR-2

10

hsa-m

iR-2

12

hsa-m

iR-

hsa-m

iR-1

7

hsa-m

iR-1

22

hsa-m

iR-1

43

hsa-m

iR-1

45

hsa-m

iR-1

06a

hsa-m

iR-

hsa-m

iR-1

0b

hsa-m

iR-1

hsa-m

iR-2

23

hsa-m

iR-1

81b

hsa-m

iR-1

55

hsa-le

t-7g

hsa-le

t-7i

hsa-m

iR-9

6

hsa-m

iR-1

37

hsa-m

iR-1

85

hsa-m

iR-1

38

hsa-m

iR-1

98

hsa-m

iR-1

33a

hsa-m

iR-1

33b

hsa-m

iR-1

5a

hsa-m

iR-3

4a

hsa-m

iR-2

9a

hsa-m

iR-2

9b

hsa-m

iR-1

03

hsa-m

iR-2

22

hsa-m

iR-1

46a

cel-m

iR-3

9cel-m

iR-5

4

cel-m

iR-2

38

Regions

Ne

t S

ign

al In

ten

sit

y (

MF

I)

106a

17


Total RNA Samples

53-Plex Assay

miR-9 miR-122 miR-1

0

5000

10000

15000

20000

25000

Samples

Ne

t S

ign

al In

ten

sit

y (

MF

I)

miR-9

miR-122

miR-1






289704

12431797

4050

7044

12899

18030

24973

23884

21852

19249

11326

6573

3852

2471

0

5000

10000

15000

20000

25000

30000


Input Amount

Ne

t S

ign

al In

ten

sit

y (

MF

I)

200 amol


81


0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000



Ne

t S

ign

al in

ten

sit

y (

MF

I)

82

miR-133a

000

020

040

060

080

100

120


Total RNA Samples

Rela

tive C

han

ge t

o H

eart

FlexmiR v2

qRT-PCR

Tissue

Re

lati

ve

Ch

an

ge


LNP-miRNA

qRT-PCR

83

miRNA

Re

lati

ve

Ch

an

ge



0

5

10

15

20

25


miRNA

Rela

tive C

han

ge

FlexmiR v2

qRT-PCR

LNP-miRNA

qPCR

84



0

5000

10000

15000

20000

25000

30000

hsa-m

iR-1

07

hsa-m

iR-1

47

hsa-m

iR-1

81a

hsa-m

iR-1

92

hsa-m

iR-1

9b

hsa-m

iR-2

20a

hsa-m

iR-2

1hsa-m

iR-2

18

hsa-le

t-7c

hsa-m

iR-1

00

hsa-m

iR-1

25b

hsa-m

iR-

hsa-m

iR-2

05

hsa-m

iR-2

21

hsa-m

iR-2

00a

hsa-m

iR-2

00c

hsa-m

iR-2

06

hsa-m

iR-1

0a

hsa-m

iR-1

6hsa-m

iR-9

hsa-m

iR-4

24

hsa-m

iR-2

0a

hsa-m

iR-2

0b

hsa-le

t-7a

hsa-m

iR-9

5hsa-m

iR-1

36

hsa-m

iR-1

95

hsa-m

iR-2

10

hsa-m

iR-2

12

hsa-m

iR-

hsa-m

iR-1

7hsa-m

iR-1

22

hsa-m

iR-1

43

hsa-m

iR-1

45

hsa-m

ir-1

06a

hsa-m

iR-

hsa-m

iR-1

0b

hsa-m

iR-1

hsa-m

iR-2

23

hsa-m

iR-1

81b

hsa-le

t-155

hsa-le

t-7g

hsa-m

iR-7

ihsa-m

iR-9

6hsa-m

iR-1

37

hsa-m

iR-1

85

hsa-m

iR-1

38

hsa-m

iR-1

33a

hsa-m

iR-1

33b

hsa-m

iR-1

5a

hsa-m

iR-3

4a

hsa-m

iR-2

9a

hsa-m

iR-2

9b

hsa-m

iR-1

03

hsa-m

iR-2

22

hsa-m

iR-1

46a

hsa-m

iR-3

9cel-m

iR-3

9cel-m

iR-2

38

Regions

Ne

t S

ign

al in

ten

sit

y (

MF

I)

50ng

100ng

250ng

500ng








bull Flexible





Proteomic Assays



Nucleic Acids








or IgM





Multiplex Detection

Detection


IgG

1 2 3 4





















Y Y

Y

IgG1 IgG2

IgG3

IgG4 IgA



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Detection



Antigen

1 2 3








Y Y Y

Y

Y

Y Y

Y Y

Y Y

Y



Y


Detection

Multiplex Detection


















compounds





compounds



SAPE SAPE














SAPE SAPE


Detection

Multiplex Detection












543-550






GTPases 827 271-282














Single Plex Assays

To Luminex

Multiplex

Assays









TAG

SAPE

Detection



Bead Hyb 80 minutes







Incubation








Targeted PCR




Upstream LDR Probes

A T

G



SNP

A

A



A


Multiple

Ligation

cycles









TAG

SAPE

Detection






Forward primers

gDNA


5rsquo

3rsquo


A

T

5rsquo

3rsquo

G





Targeted PCR



A

5rsquo

3rsquo

G

5rsquo

3rsquo

T




A

5rsquo

3rsquo

G 5rsquo

3rsquo

T








xTAG


Detection

SAPE










xTAG primer

gDNA


Biotinylated primer

5rsquo 3rsquo

3rsquo

dATP dTTP dGTP dCTP

= Carbon Spacer











Detection

Sequence Detection

TAG


















Workflow

75



2 hours



30 minutes



30 minutes





30 minutes

76






0

500

1000

1500

2000

2500

3000

3500

4000


Sample

Sig

nal In

ten

sit

y (

MF

I)

let-7c

Let-7a

77


miR-107 vs miR-103

0

1000

2000

3000

4000

5000

6000


Sample

Sig

na

l In

ten

sit

y (

MF

I)

miR-103

miR-107









0

1000

2000

3000

4000

5000

6000

7000

8000

9000

10000

hsa-m

iR-1

07

hsa-m

iR-1

47

hsa-m

iR-1

81a

hsa-m

iR-1

92

hsa-m

iR-1

9b

hsa-m

iR-2

20a

hsa-m

iR-2

1hsa-m

iR-2

18

hsa-le

t-7c

hsa-m

iR-1

00

hsa-m

iR-1

25b

hsa-m

iR-

hsa-m

iR-2

05

hsa-m

iR-2

21

hsa-m

iR-2

00a

hsa-m

iR-2

00c

hsa-m

iR-2

06

hsa-m

iR-1

0a

hsa-m

iR-1

6

hsa-m

iR-9

hsa-m

iR-4

24

hsa-m

iR-2

0a

hsa-m

iR-2

0b

hsa-le

t-7a

hsa-m

iR-9

5hsa-m

iR-1

36

hsa-m

iR-1

95

hsa-m

iR-2

10

hsa-m

iR-2

12

hsa-m

iR-

hsa-m

iR-1

7

hsa-m

iR-1

22

hsa-m

iR-1

43

hsa-m

iR-1

45

hsa-m

iR-1

06a

hsa-m

iR-

hsa-m

iR-1

0b

hsa-m

iR-1

hsa-m

iR-2

23

hsa-m

iR-1

81b

hsa-m

iR-1

55

hsa-le

t-7g

hsa-le

t-7i

hsa-m

iR-9

6

hsa-m

iR-1

37

hsa-m

iR-1

85

hsa-m

iR-1

38

hsa-m

iR-1

98

hsa-m

iR-1

33a

hsa-m

iR-1

33b

hsa-m

iR-1

5a

hsa-m

iR-3

4a

hsa-m

iR-2

9a

hsa-m

iR-2

9b

hsa-m

iR-1

03

hsa-m

iR-2

22

hsa-m

iR-1

46a

cel-m

iR-3

9cel-m

iR-5

4

cel-m

iR-2

38

Regions

Ne

t S

ign

al In

ten

sit

y (

MF

I)

106a

17


Total RNA Samples

53-Plex Assay

miR-9 miR-122 miR-1

0

5000

10000

15000

20000

25000

Samples

Ne

t S

ign

al In

ten

sit

y (

MF

I)

miR-9

miR-122

miR-1






289704

12431797

4050

7044

12899

18030

24973

23884

21852

19249

11326

6573

3852

2471

0

5000

10000

15000

20000

25000

30000


Input Amount

Ne

t S

ign

al In

ten

sit

y (

MF

I)

200 amol


81


0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000



Ne

t S

ign

al in

ten

sit

y (

MF

I)

82

miR-133a

000

020

040

060

080

100

120


Total RNA Samples

Rela

tive C

han

ge t

o H

eart

FlexmiR v2

qRT-PCR

Tissue

Re

lati

ve

Ch

an

ge


LNP-miRNA

qRT-PCR

83

miRNA

Re

lati

ve

Ch

an

ge



0

5

10

15

20

25


miRNA

Rela

tive C

han

ge

FlexmiR v2

qRT-PCR

LNP-miRNA

qPCR

84



0

5000

10000

15000

20000

25000

30000

hsa-m

iR-1

07

hsa-m

iR-1

47

hsa-m

iR-1

81a

hsa-m

iR-1

92

hsa-m

iR-1

9b

hsa-m

iR-2

20a

hsa-m

iR-2

1hsa-m

iR-2

18

hsa-le

t-7c

hsa-m

iR-1

00

hsa-m

iR-1

25b

hsa-m

iR-

hsa-m

iR-2

05

hsa-m

iR-2

21

hsa-m

iR-2

00a

hsa-m

iR-2

00c

hsa-m

iR-2

06

hsa-m

iR-1

0a

hsa-m

iR-1

6hsa-m

iR-9

hsa-m

iR-4

24

hsa-m

iR-2

0a

hsa-m

iR-2

0b

hsa-le

t-7a

hsa-m

iR-9

5hsa-m

iR-1

36

hsa-m

iR-1

95

hsa-m

iR-2

10

hsa-m

iR-2

12

hsa-m

iR-

hsa-m

iR-1

7hsa-m

iR-1

22

hsa-m

iR-1

43

hsa-m

iR-1

45

hsa-m

ir-1

06a

hsa-m

iR-

hsa-m

iR-1

0b

hsa-m

iR-1

hsa-m

iR-2

23

hsa-m

iR-1

81b

hsa-le

t-155

hsa-le

t-7g

hsa-m

iR-7

ihsa-m

iR-9

6hsa-m

iR-1

37

hsa-m

iR-1

85

hsa-m

iR-1

38

hsa-m

iR-1

33a

hsa-m

iR-1

33b

hsa-m

iR-1

5a

hsa-m

iR-3

4a

hsa-m

iR-2

9a

hsa-m

iR-2

9b

hsa-m

iR-1

03

hsa-m

iR-2

22

hsa-m

iR-1

46a

hsa-m

iR-3

9cel-m

iR-3

9cel-m

iR-2

38

Regions

Ne

t S

ign

al in

ten

sit

y (

MF

I)

50ng

100ng

250ng

500ng








bull Flexible





Proteomic Assays



Nucleic Acids








or IgM





Multiplex Detection

Detection


IgG

1 2 3 4





















Y Y

Y

IgG1 IgG2

IgG3

IgG4 IgA



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Detection



Antigen

1 2 3








Y Y Y

Y

Y

Y Y

Y Y

Y Y

Y



Y


Detection

Multiplex Detection


















compounds





compounds



SAPE SAPE














SAPE SAPE


Detection

Multiplex Detection












543-550






GTPases 827 271-282














Single Plex Assays

To Luminex

Multiplex

Assays





Bead Hyb 80 minutes







Incubation








Targeted PCR




Upstream LDR Probes

A T

G



SNP

A

A



A


Multiple

Ligation

cycles









TAG

SAPE

Detection






Forward primers

gDNA


5rsquo

3rsquo


A

T

5rsquo

3rsquo

G





Targeted PCR



A

5rsquo

3rsquo

G

5rsquo

3rsquo

T




A

5rsquo

3rsquo

G 5rsquo

3rsquo

T








xTAG


Detection

SAPE










xTAG primer

gDNA


Biotinylated primer

5rsquo 3rsquo

3rsquo

dATP dTTP dGTP dCTP

= Carbon Spacer











Detection

Sequence Detection

TAG


















Workflow

75



2 hours



30 minutes



30 minutes





30 minutes

76






0

500

1000

1500

2000

2500

3000

3500

4000


Sample

Sig

nal In

ten

sit

y (

MF

I)

let-7c

Let-7a

77


miR-107 vs miR-103

0

1000

2000

3000

4000

5000

6000


Sample

Sig

na

l In

ten

sit

y (

MF

I)

miR-103

miR-107









0

1000

2000

3000

4000

5000

6000

7000

8000

9000

10000

hsa-m

iR-1

07

hsa-m

iR-1

47

hsa-m

iR-1

81a

hsa-m

iR-1

92

hsa-m

iR-1

9b

hsa-m

iR-2

20a

hsa-m

iR-2

1hsa-m

iR-2

18

hsa-le

t-7c

hsa-m

iR-1

00

hsa-m

iR-1

25b

hsa-m

iR-

hsa-m

iR-2

05

hsa-m

iR-2

21

hsa-m

iR-2

00a

hsa-m

iR-2

00c

hsa-m

iR-2

06

hsa-m

iR-1

0a

hsa-m

iR-1

6

hsa-m

iR-9

hsa-m

iR-4

24

hsa-m

iR-2

0a

hsa-m

iR-2

0b

hsa-le

t-7a

hsa-m

iR-9

5hsa-m

iR-1

36

hsa-m

iR-1

95

hsa-m

iR-2

10

hsa-m

iR-2

12

hsa-m

iR-

hsa-m

iR-1

7

hsa-m

iR-1

22

hsa-m

iR-1

43

hsa-m

iR-1

45

hsa-m

iR-1

06a

hsa-m

iR-

hsa-m

iR-1

0b

hsa-m

iR-1

hsa-m

iR-2

23

hsa-m

iR-1

81b

hsa-m

iR-1

55

hsa-le

t-7g

hsa-le

t-7i

hsa-m

iR-9

6

hsa-m

iR-1

37

hsa-m

iR-1

85

hsa-m

iR-1

38

hsa-m

iR-1

98

hsa-m

iR-1

33a

hsa-m

iR-1

33b

hsa-m

iR-1

5a

hsa-m

iR-3

4a

hsa-m

iR-2

9a

hsa-m

iR-2

9b

hsa-m

iR-1

03

hsa-m

iR-2

22

hsa-m

iR-1

46a

cel-m

iR-3

9cel-m

iR-5

4

cel-m

iR-2

38

Regions

Ne

t S

ign

al In

ten

sit

y (

MF

I)

106a

17


Total RNA Samples

53-Plex Assay

miR-9 miR-122 miR-1

0

5000

10000

15000

20000

25000

Samples

Ne

t S

ign

al In

ten

sit

y (

MF

I)

miR-9

miR-122

miR-1






289704

12431797

4050

7044

12899

18030

24973

23884

21852

19249

11326

6573

3852

2471

0

5000

10000

15000

20000

25000

30000


Input Amount

Ne

t S

ign

al In

ten

sit

y (

MF

I)

200 amol


81


0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000



Ne

t S

ign

al in

ten

sit

y (

MF

I)

82

miR-133a

000

020

040

060

080

100

120


Total RNA Samples

Rela

tive C

han

ge t

o H

eart

FlexmiR v2

qRT-PCR

Tissue

Re

lati

ve

Ch

an

ge


LNP-miRNA

qRT-PCR

83

miRNA

Re

lati

ve

Ch

an

ge



0

5

10

15

20

25


miRNA

Rela

tive C

han

ge

FlexmiR v2

qRT-PCR

LNP-miRNA

qPCR

84



0

5000

10000

15000

20000

25000

30000

hsa-m

iR-1

07

hsa-m

iR-1

47

hsa-m

iR-1

81a

hsa-m

iR-1

92

hsa-m

iR-1

9b

hsa-m

iR-2

20a

hsa-m

iR-2

1hsa-m

iR-2

18

hsa-le

t-7c

hsa-m

iR-1

00

hsa-m

iR-1

25b

hsa-m

iR-

hsa-m

iR-2

05

hsa-m

iR-2

21

hsa-m

iR-2

00a

hsa-m

iR-2

00c

hsa-m

iR-2

06

hsa-m

iR-1

0a

hsa-m

iR-1

6hsa-m

iR-9

hsa-m

iR-4

24

hsa-m

iR-2

0a

hsa-m

iR-2

0b

hsa-le

t-7a

hsa-m

iR-9

5hsa-m

iR-1

36

hsa-m

iR-1

95

hsa-m

iR-2

10

hsa-m

iR-2

12

hsa-m

iR-

hsa-m

iR-1

7hsa-m

iR-1

22

hsa-m

iR-1

43

hsa-m

iR-1

45

hsa-m

ir-1

06a

hsa-m

iR-

hsa-m

iR-1

0b

hsa-m

iR-1

hsa-m

iR-2

23

hsa-m

iR-1

81b

hsa-le

t-155

hsa-le

t-7g

hsa-m

iR-7

ihsa-m

iR-9

6hsa-m

iR-1

37

hsa-m

iR-1

85

hsa-m

iR-1

38

hsa-m

iR-1

33a

hsa-m

iR-1

33b

hsa-m

iR-1

5a

hsa-m

iR-3

4a

hsa-m

iR-2

9a

hsa-m

iR-2

9b

hsa-m

iR-1

03

hsa-m

iR-2

22

hsa-m

iR-1

46a

hsa-m

iR-3

9cel-m

iR-3

9cel-m

iR-2

38

Regions

Ne

t S

ign

al in

ten

sit

y (

MF

I)

50ng

100ng

250ng

500ng








bull Flexible





Proteomic Assays



Nucleic Acids








or IgM





Multiplex Detection

Detection


IgG

1 2 3 4





















Y Y

Y

IgG1 IgG2

IgG3

IgG4 IgA



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Detection



Antigen

1 2 3








Y Y Y

Y

Y

Y Y

Y Y

Y Y

Y



Y


Detection

Multiplex Detection


















compounds





compounds



SAPE SAPE














SAPE SAPE


Detection

Multiplex Detection












543-550






GTPases 827 271-282














Single Plex Assays

To Luminex

Multiplex

Assays










Targeted PCR




Upstream LDR Probes

A T

G



SNP

A

A



A


Multiple

Ligation

cycles









TAG

SAPE

Detection






Forward primers

gDNA


5rsquo

3rsquo


A

T

5rsquo

3rsquo

G





Targeted PCR



A

5rsquo

3rsquo

G

5rsquo

3rsquo

T




A

5rsquo

3rsquo

G 5rsquo

3rsquo

T








xTAG


Detection

SAPE










xTAG primer

gDNA


Biotinylated primer

5rsquo 3rsquo

3rsquo

dATP dTTP dGTP dCTP

= Carbon Spacer











Detection

Sequence Detection

TAG


















Workflow

75



2 hours



30 minutes



30 minutes





30 minutes

76






0

500

1000

1500

2000

2500

3000

3500

4000


Sample

Sig

nal In

ten

sit

y (

MF

I)

let-7c

Let-7a

77


miR-107 vs miR-103

0

1000

2000

3000

4000

5000

6000


Sample

Sig

na

l In

ten

sit

y (

MF

I)

miR-103

miR-107









0

1000

2000

3000

4000

5000

6000

7000

8000

9000

10000

hsa-m

iR-1

07

hsa-m

iR-1

47

hsa-m

iR-1

81a

hsa-m

iR-1

92

hsa-m

iR-1

9b

hsa-m

iR-2

20a

hsa-m

iR-2

1hsa-m

iR-2

18

hsa-le

t-7c

hsa-m

iR-1

00

hsa-m

iR-1

25b

hsa-m

iR-

hsa-m

iR-2

05

hsa-m

iR-2

21

hsa-m

iR-2

00a

hsa-m

iR-2

00c

hsa-m

iR-2

06

hsa-m

iR-1

0a

hsa-m

iR-1

6

hsa-m

iR-9

hsa-m

iR-4

24

hsa-m

iR-2

0a

hsa-m

iR-2

0b

hsa-le

t-7a

hsa-m

iR-9

5hsa-m

iR-1

36

hsa-m

iR-1

95

hsa-m

iR-2

10

hsa-m

iR-2

12

hsa-m

iR-

hsa-m

iR-1

7

hsa-m

iR-1

22

hsa-m

iR-1

43

hsa-m

iR-1

45

hsa-m

iR-1

06a

hsa-m

iR-

hsa-m

iR-1

0b

hsa-m

iR-1

hsa-m

iR-2

23

hsa-m

iR-1

81b

hsa-m

iR-1

55

hsa-le

t-7g

hsa-le

t-7i

hsa-m

iR-9

6

hsa-m

iR-1

37

hsa-m

iR-1

85

hsa-m

iR-1

38

hsa-m

iR-1

98

hsa-m

iR-1

33a

hsa-m

iR-1

33b

hsa-m

iR-1

5a

hsa-m

iR-3

4a

hsa-m

iR-2

9a

hsa-m

iR-2

9b

hsa-m

iR-1

03

hsa-m

iR-2

22

hsa-m

iR-1

46a

cel-m

iR-3

9cel-m

iR-5

4

cel-m

iR-2

38

Regions

Ne

t S

ign

al In

ten

sit

y (

MF

I)

106a

17


Total RNA Samples

53-Plex Assay

miR-9 miR-122 miR-1

0

5000

10000

15000

20000

25000

Samples

Ne

t S

ign

al In

ten

sit

y (

MF

I)

miR-9

miR-122

miR-1






289704

12431797

4050

7044

12899

18030

24973

23884

21852

19249

11326

6573

3852

2471

0

5000

10000

15000

20000

25000

30000


Input Amount

Ne

t S

ign

al In

ten

sit

y (

MF

I)

200 amol


81


0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000



Ne

t S

ign

al in

ten

sit

y (

MF

I)

82

miR-133a

000

020

040

060

080

100

120


Total RNA Samples

Rela

tive C

han

ge t

o H

eart

FlexmiR v2

qRT-PCR

Tissue

Re

lati

ve

Ch

an

ge


LNP-miRNA

qRT-PCR

83

miRNA

Re

lati

ve

Ch

an

ge



0

5

10

15

20

25


miRNA

Rela

tive C

han

ge

FlexmiR v2

qRT-PCR

LNP-miRNA

qPCR

84



0

5000

10000

15000

20000

25000

30000

hsa-m

iR-1

07

hsa-m

iR-1

47

hsa-m

iR-1

81a

hsa-m

iR-1

92

hsa-m

iR-1

9b

hsa-m

iR-2

20a

hsa-m

iR-2

1hsa-m

iR-2

18

hsa-le

t-7c

hsa-m

iR-1

00

hsa-m

iR-1

25b

hsa-m

iR-

hsa-m

iR-2

05

hsa-m

iR-2

21

hsa-m

iR-2

00a

hsa-m

iR-2

00c

hsa-m

iR-2

06

hsa-m

iR-1

0a

hsa-m

iR-1

6hsa-m

iR-9

hsa-m

iR-4

24

hsa-m

iR-2

0a

hsa-m

iR-2

0b

hsa-le

t-7a

hsa-m

iR-9

5hsa-m

iR-1

36

hsa-m

iR-1

95

hsa-m

iR-2

10

hsa-m

iR-2

12

hsa-m

iR-

hsa-m

iR-1

7hsa-m

iR-1

22

hsa-m

iR-1

43

hsa-m

iR-1

45

hsa-m

ir-1

06a

hsa-m

iR-

hsa-m

iR-1

0b

hsa-m

iR-1

hsa-m

iR-2

23

hsa-m

iR-1

81b

hsa-le

t-155

hsa-le

t-7g

hsa-m

iR-7

ihsa-m

iR-9

6hsa-m

iR-1

37

hsa-m

iR-1

85

hsa-m

iR-1

38

hsa-m

iR-1

33a

hsa-m

iR-1

33b

hsa-m

iR-1

5a

hsa-m

iR-3

4a

hsa-m

iR-2

9a

hsa-m

iR-2

9b

hsa-m

iR-1

03

hsa-m

iR-2

22

hsa-m

iR-1

46a

hsa-m

iR-3

9cel-m

iR-3

9cel-m

iR-2

38

Regions

Ne

t S

ign

al in

ten

sit

y (

MF

I)

50ng

100ng

250ng

500ng








bull Flexible





Proteomic Assays



Nucleic Acids








or IgM





Multiplex Detection

Detection


IgG

1 2 3 4





















Y Y

Y

IgG1 IgG2

IgG3

IgG4 IgA



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Detection



Antigen

1 2 3








Y Y Y

Y

Y

Y Y

Y Y

Y Y

Y



Y


Detection

Multiplex Detection


















compounds





compounds



SAPE SAPE














SAPE SAPE


Detection

Multiplex Detection












543-550






GTPases 827 271-282














Single Plex Assays

To Luminex

Multiplex

Assays







Targeted PCR




Upstream LDR Probes

A T

G



SNP

A

A



A


Multiple

Ligation

cycles









TAG

SAPE

Detection






Forward primers

gDNA


5rsquo

3rsquo


A

T

5rsquo

3rsquo

G





Targeted PCR



A

5rsquo

3rsquo

G

5rsquo

3rsquo

T




A

5rsquo

3rsquo

G 5rsquo

3rsquo

T








xTAG


Detection

SAPE










xTAG primer

gDNA


Biotinylated primer

5rsquo 3rsquo

3rsquo

dATP dTTP dGTP dCTP

= Carbon Spacer











Detection

Sequence Detection

TAG


















Workflow

75



2 hours



30 minutes



30 minutes





30 minutes

76






0

500

1000

1500

2000

2500

3000

3500

4000


Sample

Sig

nal In

ten

sit

y (

MF

I)

let-7c

Let-7a

77


miR-107 vs miR-103

0

1000

2000

3000

4000

5000

6000


Sample

Sig

na

l In

ten

sit

y (

MF

I)

miR-103

miR-107









0

1000

2000

3000

4000

5000

6000

7000

8000

9000

10000

hsa-m

iR-1

07

hsa-m

iR-1

47

hsa-m

iR-1

81a

hsa-m

iR-1

92

hsa-m

iR-1

9b

hsa-m

iR-2

20a

hsa-m

iR-2

1hsa-m

iR-2

18

hsa-le

t-7c

hsa-m

iR-1

00

hsa-m

iR-1

25b

hsa-m

iR-

hsa-m

iR-2

05

hsa-m

iR-2

21

hsa-m

iR-2

00a

hsa-m

iR-2

00c

hsa-m

iR-2

06

hsa-m

iR-1

0a

hsa-m

iR-1

6

hsa-m

iR-9

hsa-m

iR-4

24

hsa-m

iR-2

0a

hsa-m

iR-2

0b

hsa-le

t-7a

hsa-m

iR-9

5hsa-m

iR-1

36

hsa-m

iR-1

95

hsa-m

iR-2

10

hsa-m

iR-2

12

hsa-m

iR-

hsa-m

iR-1

7

hsa-m

iR-1

22

hsa-m

iR-1

43

hsa-m

iR-1

45

hsa-m

iR-1

06a

hsa-m

iR-

hsa-m

iR-1

0b

hsa-m

iR-1

hsa-m

iR-2

23

hsa-m

iR-1

81b

hsa-m

iR-1

55

hsa-le

t-7g

hsa-le

t-7i

hsa-m

iR-9

6

hsa-m

iR-1

37

hsa-m

iR-1

85

hsa-m

iR-1

38

hsa-m

iR-1

98

hsa-m

iR-1

33a

hsa-m

iR-1

33b

hsa-m

iR-1

5a

hsa-m

iR-3

4a

hsa-m

iR-2

9a

hsa-m

iR-2

9b

hsa-m

iR-1

03

hsa-m

iR-2

22

hsa-m

iR-1

46a

cel-m

iR-3

9cel-m

iR-5

4

cel-m

iR-2

38

Regions

Ne

t S

ign

al In

ten

sit

y (

MF

I)

106a

17


Total RNA Samples

53-Plex Assay

miR-9 miR-122 miR-1

0

5000

10000

15000

20000

25000

Samples

Ne

t S

ign

al In

ten

sit

y (

MF

I)

miR-9

miR-122

miR-1






289704

12431797

4050

7044

12899

18030

24973

23884

21852

19249

11326

6573

3852

2471

0

5000

10000

15000

20000

25000

30000


Input Amount

Ne

t S

ign

al In

ten

sit

y (

MF

I)

200 amol


81


0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000



Ne

t S

ign

al in

ten

sit

y (

MF

I)

82

miR-133a

000

020

040

060

080

100

120


Total RNA Samples

Rela

tive C

han

ge t

o H

eart

FlexmiR v2

qRT-PCR

Tissue

Re

lati

ve

Ch

an

ge


LNP-miRNA

qRT-PCR

83

miRNA

Re

lati

ve

Ch

an

ge



0

5

10

15

20

25


miRNA

Rela

tive C

han

ge

FlexmiR v2

qRT-PCR

LNP-miRNA

qPCR

84



0

5000

10000

15000

20000

25000

30000

hsa-m

iR-1

07

hsa-m

iR-1

47

hsa-m

iR-1

81a

hsa-m

iR-1

92

hsa-m

iR-1

9b

hsa-m

iR-2

20a

hsa-m

iR-2

1hsa-m

iR-2

18

hsa-le

t-7c

hsa-m

iR-1

00

hsa-m

iR-1

25b

hsa-m

iR-

hsa-m

iR-2

05

hsa-m

iR-2

21

hsa-m

iR-2

00a

hsa-m

iR-2

00c

hsa-m

iR-2

06

hsa-m

iR-1

0a

hsa-m

iR-1

6hsa-m

iR-9

hsa-m

iR-4

24

hsa-m

iR-2

0a

hsa-m

iR-2

0b

hsa-le

t-7a

hsa-m

iR-9

5hsa-m

iR-1

36

hsa-m

iR-1

95

hsa-m

iR-2

10

hsa-m

iR-2

12

hsa-m

iR-

hsa-m

iR-1

7hsa-m

iR-1

22

hsa-m

iR-1

43

hsa-m

iR-1

45

hsa-m

ir-1

06a

hsa-m

iR-

hsa-m

iR-1

0b

hsa-m

iR-1

hsa-m

iR-2

23

hsa-m

iR-1

81b

hsa-le

t-155

hsa-le

t-7g

hsa-m

iR-7

ihsa-m

iR-9

6hsa-m

iR-1

37

hsa-m

iR-1

85

hsa-m

iR-1

38

hsa-m

iR-1

33a

hsa-m

iR-1

33b

hsa-m

iR-1

5a

hsa-m

iR-3

4a

hsa-m

iR-2

9a

hsa-m

iR-2

9b

hsa-m

iR-1

03

hsa-m

iR-2

22

hsa-m

iR-1

46a

hsa-m

iR-3

9cel-m

iR-3

9cel-m

iR-2

38

Regions

Ne

t S

ign

al in

ten

sit

y (

MF

I)

50ng

100ng

250ng

500ng








bull Flexible





Proteomic Assays



Nucleic Acids








or IgM





Multiplex Detection

Detection


IgG

1 2 3 4





















Y Y

Y

IgG1 IgG2

IgG3

IgG4 IgA



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Detection



Antigen

1 2 3








Y Y Y

Y

Y

Y Y

Y Y

Y Y

Y



Y


Detection

Multiplex Detection


















compounds





compounds



SAPE SAPE














SAPE SAPE


Detection

Multiplex Detection












543-550






GTPases 827 271-282














Single Plex Assays

To Luminex

Multiplex

Assays






Targeted PCR




Upstream LDR Probes

A T

G



SNP

A

A



A


Multiple

Ligation

cycles









TAG

SAPE

Detection






Forward primers

gDNA


5rsquo

3rsquo


A

T

5rsquo

3rsquo

G





Targeted PCR



A

5rsquo

3rsquo

G

5rsquo

3rsquo

T




A

5rsquo

3rsquo

G 5rsquo

3rsquo

T








xTAG


Detection

SAPE










xTAG primer

gDNA


Biotinylated primer

5rsquo 3rsquo

3rsquo

dATP dTTP dGTP dCTP

= Carbon Spacer











Detection

Sequence Detection

TAG


















Workflow

75



2 hours



30 minutes



30 minutes





30 minutes

76






0

500

1000

1500

2000

2500

3000

3500

4000


Sample

Sig

nal In

ten

sit

y (

MF

I)

let-7c

Let-7a

77


miR-107 vs miR-103

0

1000

2000

3000

4000

5000

6000


Sample

Sig

na

l In

ten

sit

y (

MF

I)

miR-103

miR-107









0

1000

2000

3000

4000

5000

6000

7000

8000

9000

10000

hsa-m

iR-1

07

hsa-m

iR-1

47

hsa-m

iR-1

81a

hsa-m

iR-1

92

hsa-m

iR-1

9b

hsa-m

iR-2

20a

hsa-m

iR-2

1hsa-m

iR-2

18

hsa-le

t-7c

hsa-m

iR-1

00

hsa-m

iR-1

25b

hsa-m

iR-

hsa-m

iR-2

05

hsa-m

iR-2

21

hsa-m

iR-2

00a

hsa-m

iR-2

00c

hsa-m

iR-2

06

hsa-m

iR-1

0a

hsa-m

iR-1

6

hsa-m

iR-9

hsa-m

iR-4

24

hsa-m

iR-2

0a

hsa-m

iR-2

0b

hsa-le

t-7a

hsa-m

iR-9

5hsa-m

iR-1

36

hsa-m

iR-1

95

hsa-m

iR-2

10

hsa-m

iR-2

12

hsa-m

iR-

hsa-m

iR-1

7

hsa-m

iR-1

22

hsa-m

iR-1

43

hsa-m

iR-1

45

hsa-m

iR-1

06a

hsa-m

iR-

hsa-m

iR-1

0b

hsa-m

iR-1

hsa-m

iR-2

23

hsa-m

iR-1

81b

hsa-m

iR-1

55

hsa-le

t-7g

hsa-le

t-7i

hsa-m

iR-9

6

hsa-m

iR-1

37

hsa-m

iR-1

85

hsa-m

iR-1

38

hsa-m

iR-1

98

hsa-m

iR-1

33a

hsa-m

iR-1

33b

hsa-m

iR-1

5a

hsa-m

iR-3

4a

hsa-m

iR-2

9a

hsa-m

iR-2

9b

hsa-m

iR-1

03

hsa-m

iR-2

22

hsa-m

iR-1

46a

cel-m

iR-3

9cel-m

iR-5

4

cel-m

iR-2

38

Regions

Ne

t S

ign

al In

ten

sit

y (

MF

I)

106a

17


Total RNA Samples

53-Plex Assay

miR-9 miR-122 miR-1

0

5000

10000

15000

20000

25000

Samples

Ne

t S

ign

al In

ten

sit

y (

MF

I)

miR-9

miR-122

miR-1






289704

12431797

4050

7044

12899

18030

24973

23884

21852

19249

11326

6573

3852

2471

0

5000

10000

15000

20000

25000

30000


Input Amount

Ne

t S

ign

al In

ten

sit

y (

MF

I)

200 amol


81


0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000



Ne

t S

ign

al in

ten

sit

y (

MF

I)

82

miR-133a

000

020

040

060

080

100

120


Total RNA Samples

Rela

tive C

han

ge t

o H

eart

FlexmiR v2

qRT-PCR

Tissue

Re

lati

ve

Ch

an

ge


LNP-miRNA

qRT-PCR

83

miRNA

Re

lati

ve

Ch

an

ge



0

5

10

15

20

25


miRNA

Rela

tive C

han

ge

FlexmiR v2

qRT-PCR

LNP-miRNA

qPCR

84



0

5000

10000

15000

20000

25000

30000

hsa-m

iR-1

07

hsa-m

iR-1

47

hsa-m

iR-1

81a

hsa-m

iR-1

92

hsa-m

iR-1

9b

hsa-m

iR-2

20a

hsa-m

iR-2

1hsa-m

iR-2

18

hsa-le

t-7c

hsa-m

iR-1

00

hsa-m

iR-1

25b

hsa-m

iR-

hsa-m

iR-2

05

hsa-m

iR-2

21

hsa-m

iR-2

00a

hsa-m

iR-2

00c

hsa-m

iR-2

06

hsa-m

iR-1

0a

hsa-m

iR-1

6hsa-m

iR-9

hsa-m

iR-4

24

hsa-m

iR-2

0a

hsa-m

iR-2

0b

hsa-le

t-7a

hsa-m

iR-9

5hsa-m

iR-1

36

hsa-m

iR-1

95

hsa-m

iR-2

10

hsa-m

iR-2

12

hsa-m

iR-

hsa-m

iR-1

7hsa-m

iR-1

22

hsa-m

iR-1

43

hsa-m

iR-1

45

hsa-m

ir-1

06a

hsa-m

iR-

hsa-m

iR-1

0b

hsa-m

iR-1

hsa-m

iR-2

23

hsa-m

iR-1

81b

hsa-le

t-155

hsa-le

t-7g

hsa-m

iR-7

ihsa-m

iR-9

6hsa-m

iR-1

37

hsa-m

iR-1

85

hsa-m

iR-1

38

hsa-m

iR-1

33a

hsa-m

iR-1

33b

hsa-m

iR-1

5a

hsa-m

iR-3

4a

hsa-m

iR-2

9a

hsa-m

iR-2

9b

hsa-m

iR-1

03

hsa-m

iR-2

22

hsa-m

iR-1

46a

hsa-m

iR-3

9cel-m

iR-3

9cel-m

iR-2

38

Regions

Ne

t S

ign

al in

ten

sit

y (

MF

I)

50ng

100ng

250ng

500ng








bull Flexible





Proteomic Assays



Nucleic Acids








or IgM





Multiplex Detection

Detection


IgG

1 2 3 4





















Y Y

Y

IgG1 IgG2

IgG3

IgG4 IgA



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Detection



Antigen

1 2 3








Y Y Y

Y

Y

Y Y

Y Y

Y Y

Y



Y


Detection

Multiplex Detection


















compounds





compounds



SAPE SAPE














SAPE SAPE


Detection

Multiplex Detection












543-550






GTPases 827 271-282














Single Plex Assays

To Luminex

Multiplex

Assays






Upstream LDR Probes

A T

G



SNP

A

A



A


Multiple

Ligation

cycles









TAG

SAPE

Detection






Forward primers

gDNA


5rsquo

3rsquo


A

T

5rsquo

3rsquo

G





Targeted PCR



A

5rsquo

3rsquo

G

5rsquo

3rsquo

T




A

5rsquo

3rsquo

G 5rsquo

3rsquo

T








xTAG


Detection

SAPE










xTAG primer

gDNA


Biotinylated primer

5rsquo 3rsquo

3rsquo

dATP dTTP dGTP dCTP

= Carbon Spacer











Detection

Sequence Detection

TAG


















Workflow

75



2 hours



30 minutes



30 minutes





30 minutes

76






0

500

1000

1500

2000

2500

3000

3500

4000


Sample

Sig

nal In

ten

sit

y (

MF

I)

let-7c

Let-7a

77


miR-107 vs miR-103

0

1000

2000

3000

4000

5000

6000


Sample

Sig

na

l In

ten

sit

y (

MF

I)

miR-103

miR-107









0

1000

2000

3000

4000

5000

6000

7000

8000

9000

10000

hsa-m

iR-1

07

hsa-m

iR-1

47

hsa-m

iR-1

81a

hsa-m

iR-1

92

hsa-m

iR-1

9b

hsa-m

iR-2

20a

hsa-m

iR-2

1hsa-m

iR-2

18

hsa-le

t-7c

hsa-m

iR-1

00

hsa-m

iR-1

25b

hsa-m

iR-

hsa-m

iR-2

05

hsa-m

iR-2

21

hsa-m

iR-2

00a

hsa-m

iR-2

00c

hsa-m

iR-2

06

hsa-m

iR-1

0a

hsa-m

iR-1

6

hsa-m

iR-9

hsa-m

iR-4

24

hsa-m

iR-2

0a

hsa-m

iR-2

0b

hsa-le

t-7a

hsa-m

iR-9

5hsa-m

iR-1

36

hsa-m

iR-1

95

hsa-m

iR-2

10

hsa-m

iR-2

12

hsa-m

iR-

hsa-m

iR-1

7

hsa-m

iR-1

22

hsa-m

iR-1

43

hsa-m

iR-1

45

hsa-m

iR-1

06a

hsa-m

iR-

hsa-m

iR-1

0b

hsa-m

iR-1

hsa-m

iR-2

23

hsa-m

iR-1

81b

hsa-m

iR-1

55

hsa-le

t-7g

hsa-le

t-7i

hsa-m

iR-9

6

hsa-m

iR-1

37

hsa-m

iR-1

85

hsa-m

iR-1

38

hsa-m

iR-1

98

hsa-m

iR-1

33a

hsa-m

iR-1

33b

hsa-m

iR-1

5a

hsa-m

iR-3

4a

hsa-m

iR-2

9a

hsa-m

iR-2

9b

hsa-m

iR-1

03

hsa-m

iR-2

22

hsa-m

iR-1

46a

cel-m

iR-3

9cel-m

iR-5

4

cel-m

iR-2

38

Regions

Ne

t S

ign

al In

ten

sit

y (

MF

I)

106a

17


Total RNA Samples

53-Plex Assay

miR-9 miR-122 miR-1

0

5000

10000

15000

20000

25000

Samples

Ne

t S

ign

al In

ten

sit

y (

MF

I)

miR-9

miR-122

miR-1






289704

12431797

4050

7044

12899

18030

24973

23884

21852

19249

11326

6573

3852

2471

0

5000

10000

15000

20000

25000

30000


Input Amount

Ne

t S

ign

al In

ten

sit

y (

MF

I)

200 amol


81


0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000



Ne

t S

ign

al in

ten

sit

y (

MF

I)

82

miR-133a

000

020

040

060

080

100

120


Total RNA Samples

Rela

tive C

han

ge t

o H

eart

FlexmiR v2

qRT-PCR

Tissue

Re

lati

ve

Ch

an

ge


LNP-miRNA

qRT-PCR

83

miRNA

Re

lati

ve

Ch

an

ge



0

5

10

15

20

25


miRNA

Rela

tive C

han

ge

FlexmiR v2

qRT-PCR

LNP-miRNA

qPCR

84



0

5000

10000

15000

20000

25000

30000

hsa-m

iR-1

07

hsa-m

iR-1

47

hsa-m

iR-1

81a

hsa-m

iR-1

92

hsa-m

iR-1

9b

hsa-m

iR-2

20a

hsa-m

iR-2

1hsa-m

iR-2

18

hsa-le

t-7c

hsa-m

iR-1

00

hsa-m

iR-1

25b

hsa-m

iR-

hsa-m

iR-2

05

hsa-m

iR-2

21

hsa-m

iR-2

00a

hsa-m

iR-2

00c

hsa-m

iR-2

06

hsa-m

iR-1

0a

hsa-m

iR-1

6hsa-m

iR-9

hsa-m

iR-4

24

hsa-m

iR-2

0a

hsa-m

iR-2

0b

hsa-le

t-7a

hsa-m

iR-9

5hsa-m

iR-1

36

hsa-m

iR-1

95

hsa-m

iR-2

10

hsa-m

iR-2

12

hsa-m

iR-

hsa-m

iR-1

7hsa-m

iR-1

22

hsa-m

iR-1

43

hsa-m

iR-1

45

hsa-m

ir-1

06a

hsa-m

iR-

hsa-m

iR-1

0b

hsa-m

iR-1

hsa-m

iR-2

23

hsa-m

iR-1

81b

hsa-le

t-155

hsa-le

t-7g

hsa-m

iR-7

ihsa-m

iR-9

6hsa-m

iR-1

37

hsa-m

iR-1

85

hsa-m

iR-1

38

hsa-m

iR-1

33a

hsa-m

iR-1

33b

hsa-m

iR-1

5a

hsa-m

iR-3

4a

hsa-m

iR-2

9a

hsa-m

iR-2

9b

hsa-m

iR-1

03

hsa-m

iR-2

22

hsa-m

iR-1

46a

hsa-m

iR-3

9cel-m

iR-3

9cel-m

iR-2

38

Regions

Ne

t S

ign

al in

ten

sit

y (

MF

I)

50ng

100ng

250ng

500ng








bull Flexible





Proteomic Assays



Nucleic Acids








or IgM





Multiplex Detection

Detection


IgG

1 2 3 4





















Y Y

Y

IgG1 IgG2

IgG3

IgG4 IgA



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Detection



Antigen

1 2 3








Y Y Y

Y

Y

Y Y

Y Y

Y Y

Y



Y


Detection

Multiplex Detection


















compounds





compounds



SAPE SAPE














SAPE SAPE


Detection

Multiplex Detection












543-550






GTPases 827 271-282














Single Plex Assays

To Luminex

Multiplex

Assays





SNP

A

A



A


Multiple

Ligation

cycles









TAG

SAPE

Detection






Forward primers

gDNA


5rsquo

3rsquo


A

T

5rsquo

3rsquo

G





Targeted PCR



A

5rsquo

3rsquo

G

5rsquo

3rsquo

T




A

5rsquo

3rsquo

G 5rsquo

3rsquo

T








xTAG


Detection

SAPE










xTAG primer

gDNA


Biotinylated primer

5rsquo 3rsquo

3rsquo

dATP dTTP dGTP dCTP

= Carbon Spacer











Detection

Sequence Detection

TAG


















Workflow

75



2 hours



30 minutes



30 minutes





30 minutes

76






0

500

1000

1500

2000

2500

3000

3500

4000


Sample

Sig

nal In

ten

sit

y (

MF

I)

let-7c

Let-7a

77


miR-107 vs miR-103

0

1000

2000

3000

4000

5000

6000


Sample

Sig

na

l In

ten

sit

y (

MF

I)

miR-103

miR-107









0

1000

2000

3000

4000

5000

6000

7000

8000

9000

10000

hsa-m

iR-1

07

hsa-m

iR-1

47

hsa-m

iR-1

81a

hsa-m

iR-1

92

hsa-m

iR-1

9b

hsa-m

iR-2

20a

hsa-m

iR-2

1hsa-m

iR-2

18

hsa-le

t-7c

hsa-m

iR-1

00

hsa-m

iR-1

25b

hsa-m

iR-

hsa-m

iR-2

05

hsa-m

iR-2

21

hsa-m

iR-2

00a

hsa-m

iR-2

00c

hsa-m

iR-2

06

hsa-m

iR-1

0a

hsa-m

iR-1

6

hsa-m

iR-9

hsa-m

iR-4

24

hsa-m

iR-2

0a

hsa-m

iR-2

0b

hsa-le

t-7a

hsa-m

iR-9

5hsa-m

iR-1

36

hsa-m

iR-1

95

hsa-m

iR-2

10

hsa-m

iR-2

12

hsa-m

iR-

hsa-m

iR-1

7

hsa-m

iR-1

22

hsa-m

iR-1

43

hsa-m

iR-1

45

hsa-m

iR-1

06a

hsa-m

iR-

hsa-m

iR-1

0b

hsa-m

iR-1

hsa-m

iR-2

23

hsa-m

iR-1

81b

hsa-m

iR-1

55

hsa-le

t-7g

hsa-le

t-7i

hsa-m

iR-9

6

hsa-m

iR-1

37

hsa-m

iR-1

85

hsa-m

iR-1

38

hsa-m

iR-1

98

hsa-m

iR-1

33a

hsa-m

iR-1

33b

hsa-m

iR-1

5a

hsa-m

iR-3

4a

hsa-m

iR-2

9a

hsa-m

iR-2

9b

hsa-m

iR-1

03

hsa-m

iR-2

22

hsa-m

iR-1

46a

cel-m

iR-3

9cel-m

iR-5

4

cel-m

iR-2

38

Regions

Ne

t S

ign

al In

ten

sit

y (

MF

I)

106a

17


Total RNA Samples

53-Plex Assay

miR-9 miR-122 miR-1

0

5000

10000

15000

20000

25000

Samples

Ne

t S

ign

al In

ten

sit

y (

MF

I)

miR-9

miR-122

miR-1






289704

12431797

4050

7044

12899

18030

24973

23884

21852

19249

11326

6573

3852

2471

0

5000

10000

15000

20000

25000

30000


Input Amount

Ne

t S

ign

al In

ten

sit

y (

MF

I)

200 amol


81


0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000



Ne

t S

ign

al in

ten

sit

y (

MF

I)

82

miR-133a

000

020

040

060

080

100

120


Total RNA Samples

Rela

tive C

han

ge t

o H

eart

FlexmiR v2

qRT-PCR

Tissue

Re

lati

ve

Ch

an

ge


LNP-miRNA

qRT-PCR

83

miRNA

Re

lati

ve

Ch

an

ge



0

5

10

15

20

25


miRNA

Rela

tive C

han

ge

FlexmiR v2

qRT-PCR

LNP-miRNA

qPCR

84



0

5000

10000

15000

20000

25000

30000

hsa-m

iR-1

07

hsa-m

iR-1

47

hsa-m

iR-1

81a

hsa-m

iR-1

92

hsa-m

iR-1

9b

hsa-m

iR-2

20a

hsa-m

iR-2

1hsa-m

iR-2

18

hsa-le

t-7c

hsa-m

iR-1

00

hsa-m

iR-1

25b

hsa-m

iR-

hsa-m

iR-2

05

hsa-m

iR-2

21

hsa-m

iR-2

00a

hsa-m

iR-2

00c

hsa-m

iR-2

06

hsa-m

iR-1

0a

hsa-m

iR-1

6hsa-m

iR-9

hsa-m

iR-4

24

hsa-m

iR-2

0a

hsa-m

iR-2

0b

hsa-le

t-7a

hsa-m

iR-9

5hsa-m

iR-1

36

hsa-m

iR-1

95

hsa-m

iR-2

10

hsa-m

iR-2

12

hsa-m

iR-

hsa-m

iR-1

7hsa-m

iR-1

22

hsa-m

iR-1

43

hsa-m

iR-1

45

hsa-m

ir-1

06a

hsa-m

iR-

hsa-m

iR-1

0b

hsa-m

iR-1

hsa-m

iR-2

23

hsa-m

iR-1

81b

hsa-le

t-155

hsa-le

t-7g

hsa-m

iR-7

ihsa-m

iR-9

6hsa-m

iR-1

37

hsa-m

iR-1

85

hsa-m

iR-1

38

hsa-m

iR-1

33a

hsa-m

iR-1

33b

hsa-m

iR-1

5a

hsa-m

iR-3

4a

hsa-m

iR-2

9a

hsa-m

iR-2

9b

hsa-m

iR-1

03

hsa-m

iR-2

22

hsa-m

iR-1

46a

hsa-m

iR-3

9cel-m

iR-3

9cel-m

iR-2

38

Regions

Ne

t S

ign

al in

ten

sit

y (

MF

I)

50ng

100ng

250ng

500ng








bull Flexible





Proteomic Assays



Nucleic Acids








or IgM





Multiplex Detection

Detection


IgG

1 2 3 4





















Y Y

Y

IgG1 IgG2

IgG3

IgG4 IgA



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Detection



Antigen

1 2 3








Y Y Y

Y

Y

Y Y

Y Y

Y Y

Y



Y


Detection

Multiplex Detection


















compounds





compounds



SAPE SAPE














SAPE SAPE


Detection

Multiplex Detection












543-550






GTPases 827 271-282














Single Plex Assays

To Luminex

Multiplex

Assays




Multiple

Ligation

cycles









TAG

SAPE

Detection






Forward primers

gDNA


5rsquo

3rsquo


A

T

5rsquo

3rsquo

G





Targeted PCR



A

5rsquo

3rsquo

G

5rsquo

3rsquo

T




A

5rsquo

3rsquo

G 5rsquo

3rsquo

T








xTAG


Detection

SAPE










xTAG primer

gDNA


Biotinylated primer

5rsquo 3rsquo

3rsquo

dATP dTTP dGTP dCTP

= Carbon Spacer











Detection

Sequence Detection

TAG


















Workflow

75



2 hours



30 minutes



30 minutes





30 minutes

76






0

500

1000

1500

2000

2500

3000

3500

4000


Sample

Sig

nal In

ten

sit

y (

MF

I)

let-7c

Let-7a

77


miR-107 vs miR-103

0

1000

2000

3000

4000

5000

6000


Sample

Sig

na

l In

ten

sit

y (

MF

I)

miR-103

miR-107









0

1000

2000

3000

4000

5000

6000

7000

8000

9000

10000

hsa-m

iR-1

07

hsa-m

iR-1

47

hsa-m

iR-1

81a

hsa-m

iR-1

92

hsa-m

iR-1

9b

hsa-m

iR-2

20a

hsa-m

iR-2

1hsa-m

iR-2

18

hsa-le

t-7c

hsa-m

iR-1

00

hsa-m

iR-1

25b

hsa-m

iR-

hsa-m

iR-2

05

hsa-m

iR-2

21

hsa-m

iR-2

00a

hsa-m

iR-2

00c

hsa-m

iR-2

06

hsa-m

iR-1

0a

hsa-m

iR-1

6

hsa-m

iR-9

hsa-m

iR-4

24

hsa-m

iR-2

0a

hsa-m

iR-2

0b

hsa-le

t-7a

hsa-m

iR-9

5hsa-m

iR-1

36

hsa-m

iR-1

95

hsa-m

iR-2

10

hsa-m

iR-2

12

hsa-m

iR-

hsa-m

iR-1

7

hsa-m

iR-1

22

hsa-m

iR-1

43

hsa-m

iR-1

45

hsa-m

iR-1

06a

hsa-m

iR-

hsa-m

iR-1

0b

hsa-m

iR-1

hsa-m

iR-2

23

hsa-m

iR-1

81b

hsa-m

iR-1

55

hsa-le

t-7g

hsa-le

t-7i

hsa-m

iR-9

6

hsa-m

iR-1

37

hsa-m

iR-1

85

hsa-m

iR-1

38

hsa-m

iR-1

98

hsa-m

iR-1

33a

hsa-m

iR-1

33b

hsa-m

iR-1

5a

hsa-m

iR-3

4a

hsa-m

iR-2

9a

hsa-m

iR-2

9b

hsa-m

iR-1

03

hsa-m

iR-2

22

hsa-m

iR-1

46a

cel-m

iR-3

9cel-m

iR-5

4

cel-m

iR-2

38

Regions

Ne

t S

ign

al In

ten

sit

y (

MF

I)

106a

17


Total RNA Samples

53-Plex Assay

miR-9 miR-122 miR-1

0

5000

10000

15000

20000

25000

Samples

Ne

t S

ign

al In

ten

sit

y (

MF

I)

miR-9

miR-122

miR-1






289704

12431797

4050

7044

12899

18030

24973

23884

21852

19249

11326

6573

3852

2471

0

5000

10000

15000

20000

25000

30000


Input Amount

Ne

t S

ign

al In

ten

sit

y (

MF

I)

200 amol


81


0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000



Ne

t S

ign

al in

ten

sit

y (

MF

I)

82

miR-133a

000

020

040

060

080

100

120


Total RNA Samples

Rela

tive C

han

ge t

o H

eart

FlexmiR v2

qRT-PCR

Tissue

Re

lati

ve

Ch

an

ge


LNP-miRNA

qRT-PCR

83

miRNA

Re

lati

ve

Ch

an

ge



0

5

10

15

20

25


miRNA

Rela

tive C

han

ge

FlexmiR v2

qRT-PCR

LNP-miRNA

qPCR

84



0

5000

10000

15000

20000

25000

30000

hsa-m

iR-1

07

hsa-m

iR-1

47

hsa-m

iR-1

81a

hsa-m

iR-1

92

hsa-m

iR-1

9b

hsa-m

iR-2

20a

hsa-m

iR-2

1hsa-m

iR-2

18

hsa-le

t-7c

hsa-m

iR-1

00

hsa-m

iR-1

25b

hsa-m

iR-

hsa-m

iR-2

05

hsa-m

iR-2

21

hsa-m

iR-2

00a

hsa-m

iR-2

00c

hsa-m

iR-2

06

hsa-m

iR-1

0a

hsa-m

iR-1

6hsa-m

iR-9

hsa-m

iR-4

24

hsa-m

iR-2

0a

hsa-m

iR-2

0b

hsa-le

t-7a

hsa-m

iR-9

5hsa-m

iR-1

36

hsa-m

iR-1

95

hsa-m

iR-2

10

hsa-m

iR-2

12

hsa-m

iR-

hsa-m

iR-1

7hsa-m

iR-1

22

hsa-m

iR-1

43

hsa-m

iR-1

45

hsa-m

ir-1

06a

hsa-m

iR-

hsa-m

iR-1

0b

hsa-m

iR-1

hsa-m

iR-2

23

hsa-m

iR-1

81b

hsa-le

t-155

hsa-le

t-7g

hsa-m

iR-7

ihsa-m

iR-9

6hsa-m

iR-1

37

hsa-m

iR-1

85

hsa-m

iR-1

38

hsa-m

iR-1

33a

hsa-m

iR-1

33b

hsa-m

iR-1

5a

hsa-m

iR-3

4a

hsa-m

iR-2

9a

hsa-m

iR-2

9b

hsa-m

iR-1

03

hsa-m

iR-2

22

hsa-m

iR-1

46a

hsa-m

iR-3

9cel-m

iR-3

9cel-m

iR-2

38

Regions

Ne

t S

ign

al in

ten

sit

y (

MF

I)

50ng

100ng

250ng

500ng








bull Flexible





Proteomic Assays



Nucleic Acids








or IgM





Multiplex Detection

Detection


IgG

1 2 3 4





















Y Y

Y

IgG1 IgG2

IgG3

IgG4 IgA



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Detection



Antigen

1 2 3








Y Y Y

Y

Y

Y Y

Y Y

Y Y

Y



Y


Detection

Multiplex Detection


















compounds





compounds



SAPE SAPE














SAPE SAPE


Detection

Multiplex Detection












543-550






GTPases 827 271-282














Single Plex Assays

To Luminex

Multiplex

Assays









TAG

SAPE

Detection






Forward primers

gDNA


5rsquo

3rsquo


A

T

5rsquo

3rsquo

G





Targeted PCR



A

5rsquo

3rsquo

G

5rsquo

3rsquo

T




A

5rsquo

3rsquo

G 5rsquo

3rsquo

T








xTAG


Detection

SAPE










xTAG primer

gDNA


Biotinylated primer

5rsquo 3rsquo

3rsquo

dATP dTTP dGTP dCTP

= Carbon Spacer











Detection

Sequence Detection

TAG


















Workflow

75



2 hours



30 minutes



30 minutes





30 minutes

76






0

500

1000

1500

2000

2500

3000

3500

4000


Sample

Sig

nal In

ten

sit

y (

MF

I)

let-7c

Let-7a

77


miR-107 vs miR-103

0

1000

2000

3000

4000

5000

6000


Sample

Sig

na

l In

ten

sit

y (

MF

I)

miR-103

miR-107









0

1000

2000

3000

4000

5000

6000

7000

8000

9000

10000

hsa-m

iR-1

07

hsa-m

iR-1

47

hsa-m

iR-1

81a

hsa-m

iR-1

92

hsa-m

iR-1

9b

hsa-m

iR-2

20a

hsa-m

iR-2

1hsa-m

iR-2

18

hsa-le

t-7c

hsa-m

iR-1

00

hsa-m

iR-1

25b

hsa-m

iR-

hsa-m

iR-2

05

hsa-m

iR-2

21

hsa-m

iR-2

00a

hsa-m

iR-2

00c

hsa-m

iR-2

06

hsa-m

iR-1

0a

hsa-m

iR-1

6

hsa-m

iR-9

hsa-m

iR-4

24

hsa-m

iR-2

0a

hsa-m

iR-2

0b

hsa-le

t-7a

hsa-m

iR-9

5hsa-m

iR-1

36

hsa-m

iR-1

95

hsa-m

iR-2

10

hsa-m

iR-2

12

hsa-m

iR-

hsa-m

iR-1

7

hsa-m

iR-1

22

hsa-m

iR-1

43

hsa-m

iR-1

45

hsa-m

iR-1

06a

hsa-m

iR-

hsa-m

iR-1

0b

hsa-m

iR-1

hsa-m

iR-2

23

hsa-m

iR-1

81b

hsa-m

iR-1

55

hsa-le

t-7g

hsa-le

t-7i

hsa-m

iR-9

6

hsa-m

iR-1

37

hsa-m

iR-1

85

hsa-m

iR-1

38

hsa-m

iR-1

98

hsa-m

iR-1

33a

hsa-m

iR-1

33b

hsa-m

iR-1

5a

hsa-m

iR-3

4a

hsa-m

iR-2

9a

hsa-m

iR-2

9b

hsa-m

iR-1

03

hsa-m

iR-2

22

hsa-m

iR-1

46a

cel-m

iR-3

9cel-m

iR-5

4

cel-m

iR-2

38

Regions

Ne

t S

ign

al In

ten

sit

y (

MF

I)

106a

17


Total RNA Samples

53-Plex Assay

miR-9 miR-122 miR-1

0

5000

10000

15000

20000

25000

Samples

Ne

t S

ign

al In

ten

sit

y (

MF

I)

miR-9

miR-122

miR-1






289704

12431797

4050

7044

12899

18030

24973

23884

21852

19249

11326

6573

3852

2471

0

5000

10000

15000

20000

25000

30000


Input Amount

Ne

t S

ign

al In

ten

sit

y (

MF

I)

200 amol


81


0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000



Ne

t S

ign

al in

ten

sit

y (

MF

I)

82

miR-133a

000

020

040

060

080

100

120


Total RNA Samples

Rela

tive C

han

ge t

o H

eart

FlexmiR v2

qRT-PCR

Tissue

Re

lati

ve

Ch

an

ge


LNP-miRNA

qRT-PCR

83

miRNA

Re

lati

ve

Ch

an

ge



0

5

10

15

20

25


miRNA

Rela

tive C

han

ge

FlexmiR v2

qRT-PCR

LNP-miRNA

qPCR

84



0

5000

10000

15000

20000

25000

30000

hsa-m

iR-1

07

hsa-m

iR-1

47

hsa-m

iR-1

81a

hsa-m

iR-1

92

hsa-m

iR-1

9b

hsa-m

iR-2

20a

hsa-m

iR-2

1hsa-m

iR-2

18

hsa-le

t-7c

hsa-m

iR-1

00

hsa-m

iR-1

25b

hsa-m

iR-

hsa-m

iR-2

05

hsa-m

iR-2

21

hsa-m

iR-2

00a

hsa-m

iR-2

00c

hsa-m

iR-2

06

hsa-m

iR-1

0a

hsa-m

iR-1

6hsa-m

iR-9

hsa-m

iR-4

24

hsa-m

iR-2

0a

hsa-m

iR-2

0b

hsa-le

t-7a

hsa-m

iR-9

5hsa-m

iR-1

36

hsa-m

iR-1

95

hsa-m

iR-2

10

hsa-m

iR-2

12

hsa-m

iR-

hsa-m

iR-1

7hsa-m

iR-1

22

hsa-m

iR-1

43

hsa-m

iR-1

45

hsa-m

ir-1

06a

hsa-m

iR-

hsa-m

iR-1

0b

hsa-m

iR-1

hsa-m

iR-2

23

hsa-m

iR-1

81b

hsa-le

t-155

hsa-le

t-7g

hsa-m

iR-7

ihsa-m

iR-9

6hsa-m

iR-1

37

hsa-m

iR-1

85

hsa-m

iR-1

38

hsa-m

iR-1

33a

hsa-m

iR-1

33b

hsa-m

iR-1

5a

hsa-m

iR-3

4a

hsa-m

iR-2

9a

hsa-m

iR-2

9b

hsa-m

iR-1

03

hsa-m

iR-2

22

hsa-m

iR-1

46a

hsa-m

iR-3

9cel-m

iR-3

9cel-m

iR-2

38

Regions

Ne

t S

ign

al in

ten

sit

y (

MF

I)

50ng

100ng

250ng

500ng








bull Flexible





Proteomic Assays



Nucleic Acids








or IgM





Multiplex Detection

Detection


IgG

1 2 3 4





















Y Y

Y

IgG1 IgG2

IgG3

IgG4 IgA



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Detection



Antigen

1 2 3








Y Y Y

Y

Y

Y Y

Y Y

Y Y

Y



Y


Detection

Multiplex Detection


















compounds





compounds



SAPE SAPE














SAPE SAPE


Detection

Multiplex Detection












543-550






GTPases 827 271-282














Single Plex Assays

To Luminex

Multiplex

Assays








Forward primers

gDNA


5rsquo

3rsquo


A

T

5rsquo

3rsquo

G





Targeted PCR



A

5rsquo

3rsquo

G

5rsquo

3rsquo

T




A

5rsquo

3rsquo

G 5rsquo

3rsquo

T








xTAG


Detection

SAPE










xTAG primer

gDNA


Biotinylated primer

5rsquo 3rsquo

3rsquo

dATP dTTP dGTP dCTP

= Carbon Spacer











Detection

Sequence Detection

TAG


















Workflow

75



2 hours



30 minutes



30 minutes





30 minutes

76






0

500

1000

1500

2000

2500

3000

3500

4000


Sample

Sig

nal In

ten

sit

y (

MF

I)

let-7c

Let-7a

77


miR-107 vs miR-103

0

1000

2000

3000

4000

5000

6000


Sample

Sig

na

l In

ten

sit

y (

MF

I)

miR-103

miR-107









0

1000

2000

3000

4000

5000

6000

7000

8000

9000

10000

hsa-m

iR-1

07

hsa-m

iR-1

47

hsa-m

iR-1

81a

hsa-m

iR-1

92

hsa-m

iR-1

9b

hsa-m

iR-2

20a

hsa-m

iR-2

1hsa-m

iR-2

18

hsa-le

t-7c

hsa-m

iR-1

00

hsa-m

iR-1

25b

hsa-m

iR-

hsa-m

iR-2

05

hsa-m

iR-2

21

hsa-m

iR-2

00a

hsa-m

iR-2

00c

hsa-m

iR-2

06

hsa-m

iR-1

0a

hsa-m

iR-1

6

hsa-m

iR-9

hsa-m

iR-4

24

hsa-m

iR-2

0a

hsa-m

iR-2

0b

hsa-le

t-7a

hsa-m

iR-9

5hsa-m

iR-1

36

hsa-m

iR-1

95

hsa-m

iR-2

10

hsa-m

iR-2

12

hsa-m

iR-

hsa-m

iR-1

7

hsa-m

iR-1

22

hsa-m

iR-1

43

hsa-m

iR-1

45

hsa-m

iR-1

06a

hsa-m

iR-

hsa-m

iR-1

0b

hsa-m

iR-1

hsa-m

iR-2

23

hsa-m

iR-1

81b

hsa-m

iR-1

55

hsa-le

t-7g

hsa-le

t-7i

hsa-m

iR-9

6

hsa-m

iR-1

37

hsa-m

iR-1

85

hsa-m

iR-1

38

hsa-m

iR-1

98

hsa-m

iR-1

33a

hsa-m

iR-1

33b

hsa-m

iR-1

5a

hsa-m

iR-3

4a

hsa-m

iR-2

9a

hsa-m

iR-2

9b

hsa-m

iR-1

03

hsa-m

iR-2

22

hsa-m

iR-1

46a

cel-m

iR-3

9cel-m

iR-5

4

cel-m

iR-2

38

Regions

Ne

t S

ign

al In

ten

sit

y (

MF

I)

106a

17


Total RNA Samples

53-Plex Assay

miR-9 miR-122 miR-1

0

5000

10000

15000

20000

25000

Samples

Ne

t S

ign

al In

ten

sit

y (

MF

I)

miR-9

miR-122

miR-1






289704

12431797

4050

7044

12899

18030

24973

23884

21852

19249

11326

6573

3852

2471

0

5000

10000

15000

20000

25000

30000


Input Amount

Ne

t S

ign

al In

ten

sit

y (

MF

I)

200 amol


81


0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000



Ne

t S

ign

al in

ten

sit

y (

MF

I)

82

miR-133a

000

020

040

060

080

100

120


Total RNA Samples

Rela

tive C

han

ge t

o H

eart

FlexmiR v2

qRT-PCR

Tissue

Re

lati

ve

Ch

an

ge


LNP-miRNA

qRT-PCR

83

miRNA

Re

lati

ve

Ch

an

ge



0

5

10

15

20

25


miRNA

Rela

tive C

han

ge

FlexmiR v2

qRT-PCR

LNP-miRNA

qPCR

84



0

5000

10000

15000

20000

25000

30000

hsa-m

iR-1

07

hsa-m

iR-1

47

hsa-m

iR-1

81a

hsa-m

iR-1

92

hsa-m

iR-1

9b

hsa-m

iR-2

20a

hsa-m

iR-2

1hsa-m

iR-2

18

hsa-le

t-7c

hsa-m

iR-1

00

hsa-m

iR-1

25b

hsa-m

iR-

hsa-m

iR-2

05

hsa-m

iR-2

21

hsa-m

iR-2

00a

hsa-m

iR-2

00c

hsa-m

iR-2

06

hsa-m

iR-1

0a

hsa-m

iR-1

6hsa-m

iR-9

hsa-m

iR-4

24

hsa-m

iR-2

0a

hsa-m

iR-2

0b

hsa-le

t-7a

hsa-m

iR-9

5hsa-m

iR-1

36

hsa-m

iR-1

95

hsa-m

iR-2

10

hsa-m

iR-2

12

hsa-m

iR-

hsa-m

iR-1

7hsa-m

iR-1

22

hsa-m

iR-1

43

hsa-m

iR-1

45

hsa-m

ir-1

06a

hsa-m

iR-

hsa-m

iR-1

0b

hsa-m

iR-1

hsa-m

iR-2

23

hsa-m

iR-1

81b

hsa-le

t-155

hsa-le

t-7g

hsa-m

iR-7

ihsa-m

iR-9

6hsa-m

iR-1

37

hsa-m

iR-1

85

hsa-m

iR-1

38

hsa-m

iR-1

33a

hsa-m

iR-1

33b

hsa-m

iR-1

5a

hsa-m

iR-3

4a

hsa-m

iR-2

9a

hsa-m

iR-2

9b

hsa-m

iR-1

03

hsa-m

iR-2

22

hsa-m

iR-1

46a

hsa-m

iR-3

9cel-m

iR-3

9cel-m

iR-2

38

Regions

Ne

t S

ign

al in

ten

sit

y (

MF

I)

50ng

100ng

250ng

500ng








bull Flexible





Proteomic Assays



Nucleic Acids








or IgM





Multiplex Detection

Detection


IgG

1 2 3 4





















Y Y

Y

IgG1 IgG2

IgG3

IgG4 IgA



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Detection



Antigen

1 2 3








Y Y Y

Y

Y

Y Y

Y Y

Y Y

Y



Y


Detection

Multiplex Detection


















compounds





compounds



SAPE SAPE














SAPE SAPE


Detection

Multiplex Detection












543-550






GTPases 827 271-282














Single Plex Assays

To Luminex

Multiplex

Assays





Forward primers

gDNA


5rsquo

3rsquo


A

T

5rsquo

3rsquo

G





Targeted PCR



A

5rsquo

3rsquo

G

5rsquo

3rsquo

T




A

5rsquo

3rsquo

G 5rsquo

3rsquo

T








xTAG


Detection

SAPE










xTAG primer

gDNA


Biotinylated primer

5rsquo 3rsquo

3rsquo

dATP dTTP dGTP dCTP

= Carbon Spacer











Detection

Sequence Detection

TAG


















Workflow

75



2 hours



30 minutes



30 minutes





30 minutes

76






0

500

1000

1500

2000

2500

3000

3500

4000


Sample

Sig

nal In

ten

sit

y (

MF

I)

let-7c

Let-7a

77


miR-107 vs miR-103

0

1000

2000

3000

4000

5000

6000


Sample

Sig

na

l In

ten

sit

y (

MF

I)

miR-103

miR-107









0

1000

2000

3000

4000

5000

6000

7000

8000

9000

10000

hsa-m

iR-1

07

hsa-m

iR-1

47

hsa-m

iR-1

81a

hsa-m

iR-1

92

hsa-m

iR-1

9b

hsa-m

iR-2

20a

hsa-m

iR-2

1hsa-m

iR-2

18

hsa-le

t-7c

hsa-m

iR-1

00

hsa-m

iR-1

25b

hsa-m

iR-

hsa-m

iR-2

05

hsa-m

iR-2

21

hsa-m

iR-2

00a

hsa-m

iR-2

00c

hsa-m

iR-2

06

hsa-m

iR-1

0a

hsa-m

iR-1

6

hsa-m

iR-9

hsa-m

iR-4

24

hsa-m

iR-2

0a

hsa-m

iR-2

0b

hsa-le

t-7a

hsa-m

iR-9

5hsa-m

iR-1

36

hsa-m

iR-1

95

hsa-m

iR-2

10

hsa-m

iR-2

12

hsa-m

iR-

hsa-m

iR-1

7

hsa-m

iR-1

22

hsa-m

iR-1

43

hsa-m

iR-1

45

hsa-m

iR-1

06a

hsa-m

iR-

hsa-m

iR-1

0b

hsa-m

iR-1

hsa-m

iR-2

23

hsa-m

iR-1

81b

hsa-m

iR-1

55

hsa-le

t-7g

hsa-le

t-7i

hsa-m

iR-9

6

hsa-m

iR-1

37

hsa-m

iR-1

85

hsa-m

iR-1

38

hsa-m

iR-1

98

hsa-m

iR-1

33a

hsa-m

iR-1

33b

hsa-m

iR-1

5a

hsa-m

iR-3

4a

hsa-m

iR-2

9a

hsa-m

iR-2

9b

hsa-m

iR-1

03

hsa-m

iR-2

22

hsa-m

iR-1

46a

cel-m

iR-3

9cel-m

iR-5

4

cel-m

iR-2

38

Regions

Ne

t S

ign

al In

ten

sit

y (

MF

I)

106a

17


Total RNA Samples

53-Plex Assay

miR-9 miR-122 miR-1

0

5000

10000

15000

20000

25000

Samples

Ne

t S

ign

al In

ten

sit

y (

MF

I)

miR-9

miR-122

miR-1






289704

12431797

4050

7044

12899

18030

24973

23884

21852

19249

11326

6573

3852

2471

0

5000

10000

15000

20000

25000

30000


Input Amount

Ne

t S

ign

al In

ten

sit

y (

MF

I)

200 amol


81


0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000



Ne

t S

ign

al in

ten

sit

y (

MF

I)

82

miR-133a

000

020

040

060

080

100

120


Total RNA Samples

Rela

tive C

han

ge t

o H

eart

FlexmiR v2

qRT-PCR

Tissue

Re

lati

ve

Ch

an

ge


LNP-miRNA

qRT-PCR

83

miRNA

Re

lati

ve

Ch

an

ge



0

5

10

15

20

25


miRNA

Rela

tive C

han

ge

FlexmiR v2

qRT-PCR

LNP-miRNA

qPCR

84



0

5000

10000

15000

20000

25000

30000

hsa-m

iR-1

07

hsa-m

iR-1

47

hsa-m

iR-1

81a

hsa-m

iR-1

92

hsa-m

iR-1

9b

hsa-m

iR-2

20a

hsa-m

iR-2

1hsa-m

iR-2

18

hsa-le

t-7c

hsa-m

iR-1

00

hsa-m

iR-1

25b

hsa-m

iR-

hsa-m

iR-2

05

hsa-m

iR-2

21

hsa-m

iR-2

00a

hsa-m

iR-2

00c

hsa-m

iR-2

06

hsa-m

iR-1

0a

hsa-m

iR-1

6hsa-m

iR-9

hsa-m

iR-4

24

hsa-m

iR-2

0a

hsa-m

iR-2

0b

hsa-le

t-7a

hsa-m

iR-9

5hsa-m

iR-1

36

hsa-m

iR-1

95

hsa-m

iR-2

10

hsa-m

iR-2

12

hsa-m

iR-

hsa-m

iR-1

7hsa-m

iR-1

22

hsa-m

iR-1

43

hsa-m

iR-1

45

hsa-m

ir-1

06a

hsa-m

iR-

hsa-m

iR-1

0b

hsa-m

iR-1

hsa-m

iR-2

23

hsa-m

iR-1

81b

hsa-le

t-155

hsa-le

t-7g

hsa-m

iR-7

ihsa-m

iR-9

6hsa-m

iR-1

37

hsa-m

iR-1

85

hsa-m

iR-1

38

hsa-m

iR-1

33a

hsa-m

iR-1

33b

hsa-m

iR-1

5a

hsa-m

iR-3

4a

hsa-m

iR-2

9a

hsa-m

iR-2

9b

hsa-m

iR-1

03

hsa-m

iR-2

22

hsa-m

iR-1

46a

hsa-m

iR-3

9cel-m

iR-3

9cel-m

iR-2

38

Regions

Ne

t S

ign

al in

ten

sit

y (

MF

I)

50ng

100ng

250ng

500ng








bull Flexible





Proteomic Assays



Nucleic Acids








or IgM





Multiplex Detection

Detection


IgG

1 2 3 4





















Y Y

Y

IgG1 IgG2

IgG3

IgG4 IgA



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Detection



Antigen

1 2 3








Y Y Y

Y

Y

Y Y

Y Y

Y Y

Y



Y


Detection

Multiplex Detection


















compounds





compounds



SAPE SAPE














SAPE SAPE


Detection

Multiplex Detection












543-550






GTPases 827 271-282














Single Plex Assays

To Luminex

Multiplex

Assays




Forward primers

gDNA


5rsquo

3rsquo


A

T

5rsquo

3rsquo

G





Targeted PCR



A

5rsquo

3rsquo

G

5rsquo

3rsquo

T




A

5rsquo

3rsquo

G 5rsquo

3rsquo

T








xTAG


Detection

SAPE










xTAG primer

gDNA


Biotinylated primer

5rsquo 3rsquo

3rsquo

dATP dTTP dGTP dCTP

= Carbon Spacer











Detection

Sequence Detection

TAG


















Workflow

75



2 hours



30 minutes



30 minutes





30 minutes

76






0

500

1000

1500

2000

2500

3000

3500

4000


Sample

Sig

nal In

ten

sit

y (

MF

I)

let-7c

Let-7a

77


miR-107 vs miR-103

0

1000

2000

3000

4000

5000

6000


Sample

Sig

na

l In

ten

sit

y (

MF

I)

miR-103

miR-107









0

1000

2000

3000

4000

5000

6000

7000

8000

9000

10000

hsa-m

iR-1

07

hsa-m

iR-1

47

hsa-m

iR-1

81a

hsa-m

iR-1

92

hsa-m

iR-1

9b

hsa-m

iR-2

20a

hsa-m

iR-2

1hsa-m

iR-2

18

hsa-le

t-7c

hsa-m

iR-1

00

hsa-m

iR-1

25b

hsa-m

iR-

hsa-m

iR-2

05

hsa-m

iR-2

21

hsa-m

iR-2

00a

hsa-m

iR-2

00c

hsa-m

iR-2

06

hsa-m

iR-1

0a

hsa-m

iR-1

6

hsa-m

iR-9

hsa-m

iR-4

24

hsa-m

iR-2

0a

hsa-m

iR-2

0b

hsa-le

t-7a

hsa-m

iR-9

5hsa-m

iR-1

36

hsa-m

iR-1

95

hsa-m

iR-2

10

hsa-m

iR-2

12

hsa-m

iR-

hsa-m

iR-1

7

hsa-m

iR-1

22

hsa-m

iR-1

43

hsa-m

iR-1

45

hsa-m

iR-1

06a

hsa-m

iR-

hsa-m

iR-1

0b

hsa-m

iR-1

hsa-m

iR-2

23

hsa-m

iR-1

81b

hsa-m

iR-1

55

hsa-le

t-7g

hsa-le

t-7i

hsa-m

iR-9

6

hsa-m

iR-1

37

hsa-m

iR-1

85

hsa-m

iR-1

38

hsa-m

iR-1

98

hsa-m

iR-1

33a

hsa-m

iR-1

33b

hsa-m

iR-1

5a

hsa-m

iR-3

4a

hsa-m

iR-2

9a

hsa-m

iR-2

9b

hsa-m

iR-1

03

hsa-m

iR-2

22

hsa-m

iR-1

46a

cel-m

iR-3

9cel-m

iR-5

4

cel-m

iR-2

38

Regions

Ne

t S

ign

al In

ten

sit

y (

MF

I)

106a

17


Total RNA Samples

53-Plex Assay

miR-9 miR-122 miR-1

0

5000

10000

15000

20000

25000

Samples

Ne

t S

ign

al In

ten

sit

y (

MF

I)

miR-9

miR-122

miR-1






289704

12431797

4050

7044

12899

18030

24973

23884

21852

19249

11326

6573

3852

2471

0

5000

10000

15000

20000

25000

30000


Input Amount

Ne

t S

ign

al In

ten

sit

y (

MF

I)

200 amol


81


0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000



Ne

t S

ign

al in

ten

sit

y (

MF

I)

82

miR-133a

000

020

040

060

080

100

120


Total RNA Samples

Rela

tive C

han

ge t

o H

eart

FlexmiR v2

qRT-PCR

Tissue

Re

lati

ve

Ch

an

ge


LNP-miRNA

qRT-PCR

83

miRNA

Re

lati

ve

Ch

an

ge



0

5

10

15

20

25


miRNA

Rela

tive C

han

ge

FlexmiR v2

qRT-PCR

LNP-miRNA

qPCR

84



0

5000

10000

15000

20000

25000

30000

hsa-m

iR-1

07

hsa-m

iR-1

47

hsa-m

iR-1

81a

hsa-m

iR-1

92

hsa-m

iR-1

9b

hsa-m

iR-2

20a

hsa-m

iR-2

1hsa-m

iR-2

18

hsa-le

t-7c

hsa-m

iR-1

00

hsa-m

iR-1

25b

hsa-m

iR-

hsa-m

iR-2

05

hsa-m

iR-2

21

hsa-m

iR-2

00a

hsa-m

iR-2

00c

hsa-m

iR-2

06

hsa-m

iR-1

0a

hsa-m

iR-1

6hsa-m

iR-9

hsa-m

iR-4

24

hsa-m

iR-2

0a

hsa-m

iR-2

0b

hsa-le

t-7a

hsa-m

iR-9

5hsa-m

iR-1

36

hsa-m

iR-1

95

hsa-m

iR-2

10

hsa-m

iR-2

12

hsa-m

iR-

hsa-m

iR-1

7hsa-m

iR-1

22

hsa-m

iR-1

43

hsa-m

iR-1

45

hsa-m

ir-1

06a

hsa-m

iR-

hsa-m

iR-1

0b

hsa-m

iR-1

hsa-m

iR-2

23

hsa-m

iR-1

81b

hsa-le

t-155

hsa-le

t-7g

hsa-m

iR-7

ihsa-m

iR-9

6hsa-m

iR-1

37

hsa-m

iR-1

85

hsa-m

iR-1

38

hsa-m

iR-1

33a

hsa-m

iR-1

33b

hsa-m

iR-1

5a

hsa-m

iR-3

4a

hsa-m

iR-2

9a

hsa-m

iR-2

9b

hsa-m

iR-1

03

hsa-m

iR-2

22

hsa-m

iR-1

46a

hsa-m

iR-3

9cel-m

iR-3

9cel-m

iR-2

38

Regions

Ne

t S

ign

al in

ten

sit

y (

MF

I)

50ng

100ng

250ng

500ng








bull Flexible





Proteomic Assays



Nucleic Acids








or IgM





Multiplex Detection

Detection


IgG

1 2 3 4





















Y Y

Y

IgG1 IgG2

IgG3

IgG4 IgA



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Detection



Antigen

1 2 3








Y Y Y

Y

Y

Y Y

Y Y

Y Y

Y



Y


Detection

Multiplex Detection


















compounds





compounds



SAPE SAPE














SAPE SAPE


Detection

Multiplex Detection












543-550






GTPases 827 271-282














Single Plex Assays

To Luminex

Multiplex

Assays






Targeted PCR



A

5rsquo

3rsquo

G

5rsquo

3rsquo

T




A

5rsquo

3rsquo

G 5rsquo

3rsquo

T








xTAG


Detection

SAPE










xTAG primer

gDNA


Biotinylated primer

5rsquo 3rsquo

3rsquo

dATP dTTP dGTP dCTP

= Carbon Spacer











Detection

Sequence Detection

TAG


















Workflow

75



2 hours



30 minutes



30 minutes





30 minutes

76






0

500

1000

1500

2000

2500

3000

3500

4000


Sample

Sig

nal In

ten

sit

y (

MF

I)

let-7c

Let-7a

77


miR-107 vs miR-103

0

1000

2000

3000

4000

5000

6000


Sample

Sig

na

l In

ten

sit

y (

MF

I)

miR-103

miR-107









0

1000

2000

3000

4000

5000

6000

7000

8000

9000

10000

hsa-m

iR-1

07

hsa-m

iR-1

47

hsa-m

iR-1

81a

hsa-m

iR-1

92

hsa-m

iR-1

9b

hsa-m

iR-2

20a

hsa-m

iR-2

1hsa-m

iR-2

18

hsa-le

t-7c

hsa-m

iR-1

00

hsa-m

iR-1

25b

hsa-m

iR-

hsa-m

iR-2

05

hsa-m

iR-2

21

hsa-m

iR-2

00a

hsa-m

iR-2

00c

hsa-m

iR-2

06

hsa-m

iR-1

0a

hsa-m

iR-1

6

hsa-m

iR-9

hsa-m

iR-4

24

hsa-m

iR-2

0a

hsa-m

iR-2

0b

hsa-le

t-7a

hsa-m

iR-9

5hsa-m

iR-1

36

hsa-m

iR-1

95

hsa-m

iR-2

10

hsa-m

iR-2

12

hsa-m

iR-

hsa-m

iR-1

7

hsa-m

iR-1

22

hsa-m

iR-1

43

hsa-m

iR-1

45

hsa-m

iR-1

06a

hsa-m

iR-

hsa-m

iR-1

0b

hsa-m

iR-1

hsa-m

iR-2

23

hsa-m

iR-1

81b

hsa-m

iR-1

55

hsa-le

t-7g

hsa-le

t-7i

hsa-m

iR-9

6

hsa-m

iR-1

37

hsa-m

iR-1

85

hsa-m

iR-1

38

hsa-m

iR-1

98

hsa-m

iR-1

33a

hsa-m

iR-1

33b

hsa-m

iR-1

5a

hsa-m

iR-3

4a

hsa-m

iR-2

9a

hsa-m

iR-2

9b

hsa-m

iR-1

03

hsa-m

iR-2

22

hsa-m

iR-1

46a

cel-m

iR-3

9cel-m

iR-5

4

cel-m

iR-2

38

Regions

Ne

t S

ign

al In

ten

sit

y (

MF

I)

106a

17


Total RNA Samples

53-Plex Assay

miR-9 miR-122 miR-1

0

5000

10000

15000

20000

25000

Samples

Ne

t S

ign

al In

ten

sit

y (

MF

I)

miR-9

miR-122

miR-1






289704

12431797

4050

7044

12899

18030

24973

23884

21852

19249

11326

6573

3852

2471

0

5000

10000

15000

20000

25000

30000


Input Amount

Ne

t S

ign

al In

ten

sit

y (

MF

I)

200 amol


81


0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000



Ne

t S

ign

al in

ten

sit

y (

MF

I)

82

miR-133a

000

020

040

060

080

100

120


Total RNA Samples

Rela

tive C

han

ge t

o H

eart

FlexmiR v2

qRT-PCR

Tissue

Re

lati

ve

Ch

an

ge


LNP-miRNA

qRT-PCR

83

miRNA

Re

lati

ve

Ch

an

ge



0

5

10

15

20

25


miRNA

Rela

tive C

han

ge

FlexmiR v2

qRT-PCR

LNP-miRNA

qPCR

84



0

5000

10000

15000

20000

25000

30000

hsa-m

iR-1

07

hsa-m

iR-1

47

hsa-m

iR-1

81a

hsa-m

iR-1

92

hsa-m

iR-1

9b

hsa-m

iR-2

20a

hsa-m

iR-2

1hsa-m

iR-2

18

hsa-le

t-7c

hsa-m

iR-1

00

hsa-m

iR-1

25b

hsa-m

iR-

hsa-m

iR-2

05

hsa-m

iR-2

21

hsa-m

iR-2

00a

hsa-m

iR-2

00c

hsa-m

iR-2

06

hsa-m

iR-1

0a

hsa-m

iR-1

6hsa-m

iR-9

hsa-m

iR-4

24

hsa-m

iR-2

0a

hsa-m

iR-2

0b

hsa-le

t-7a

hsa-m

iR-9

5hsa-m

iR-1

36

hsa-m

iR-1

95

hsa-m

iR-2

10

hsa-m

iR-2

12

hsa-m

iR-

hsa-m

iR-1

7hsa-m

iR-1

22

hsa-m

iR-1

43

hsa-m

iR-1

45

hsa-m

ir-1

06a

hsa-m

iR-

hsa-m

iR-1

0b

hsa-m

iR-1

hsa-m

iR-2

23

hsa-m

iR-1

81b

hsa-le

t-155

hsa-le

t-7g

hsa-m

iR-7

ihsa-m

iR-9

6hsa-m

iR-1

37

hsa-m

iR-1

85

hsa-m

iR-1

38

hsa-m

iR-1

33a

hsa-m

iR-1

33b

hsa-m

iR-1

5a

hsa-m

iR-3

4a

hsa-m

iR-2

9a

hsa-m

iR-2

9b

hsa-m

iR-1

03

hsa-m

iR-2

22

hsa-m

iR-1

46a

hsa-m

iR-3

9cel-m

iR-3

9cel-m

iR-2

38

Regions

Ne

t S

ign

al in

ten

sit

y (

MF

I)

50ng

100ng

250ng

500ng








bull Flexible





Proteomic Assays



Nucleic Acids








or IgM





Multiplex Detection

Detection


IgG

1 2 3 4





















Y Y

Y

IgG1 IgG2

IgG3

IgG4 IgA



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Detection



Antigen

1 2 3








Y Y Y

Y

Y

Y Y

Y Y

Y Y

Y



Y


Detection

Multiplex Detection


















compounds





compounds



SAPE SAPE














SAPE SAPE


Detection

Multiplex Detection












543-550






GTPases 827 271-282














Single Plex Assays

To Luminex

Multiplex

Assays





A

5rsquo

3rsquo

G

5rsquo

3rsquo

T




A

5rsquo

3rsquo

G 5rsquo

3rsquo

T








xTAG


Detection

SAPE










xTAG primer

gDNA


Biotinylated primer

5rsquo 3rsquo

3rsquo

dATP dTTP dGTP dCTP

= Carbon Spacer











Detection

Sequence Detection

TAG


















Workflow

75



2 hours



30 minutes



30 minutes





30 minutes

76






0

500

1000

1500

2000

2500

3000

3500

4000


Sample

Sig

nal In

ten

sit

y (

MF

I)

let-7c

Let-7a

77


miR-107 vs miR-103

0

1000

2000

3000

4000

5000

6000


Sample

Sig

na

l In

ten

sit

y (

MF

I)

miR-103

miR-107









0

1000

2000

3000

4000

5000

6000

7000

8000

9000

10000

hsa-m

iR-1

07

hsa-m

iR-1

47

hsa-m

iR-1

81a

hsa-m

iR-1

92

hsa-m

iR-1

9b

hsa-m

iR-2

20a

hsa-m

iR-2

1hsa-m

iR-2

18

hsa-le

t-7c

hsa-m

iR-1

00

hsa-m

iR-1

25b

hsa-m

iR-

hsa-m

iR-2

05

hsa-m

iR-2

21

hsa-m

iR-2

00a

hsa-m

iR-2

00c

hsa-m

iR-2

06

hsa-m

iR-1

0a

hsa-m

iR-1

6

hsa-m

iR-9

hsa-m

iR-4

24

hsa-m

iR-2

0a

hsa-m

iR-2

0b

hsa-le

t-7a

hsa-m

iR-9

5hsa-m

iR-1

36

hsa-m

iR-1

95

hsa-m

iR-2

10

hsa-m

iR-2

12

hsa-m

iR-

hsa-m

iR-1

7

hsa-m

iR-1

22

hsa-m

iR-1

43

hsa-m

iR-1

45

hsa-m

iR-1

06a

hsa-m

iR-

hsa-m

iR-1

0b

hsa-m

iR-1

hsa-m

iR-2

23

hsa-m

iR-1

81b

hsa-m

iR-1

55

hsa-le

t-7g

hsa-le

t-7i

hsa-m

iR-9

6

hsa-m

iR-1

37

hsa-m

iR-1

85

hsa-m

iR-1

38

hsa-m

iR-1

98

hsa-m

iR-1

33a

hsa-m

iR-1

33b

hsa-m

iR-1

5a

hsa-m

iR-3

4a

hsa-m

iR-2

9a

hsa-m

iR-2

9b

hsa-m

iR-1

03

hsa-m

iR-2

22

hsa-m

iR-1

46a

cel-m

iR-3

9cel-m

iR-5

4

cel-m

iR-2

38

Regions

Ne

t S

ign

al In

ten

sit

y (

MF

I)

106a

17


Total RNA Samples

53-Plex Assay

miR-9 miR-122 miR-1

0

5000

10000

15000

20000

25000

Samples

Ne

t S

ign

al In

ten

sit

y (

MF

I)

miR-9

miR-122

miR-1






289704

12431797

4050

7044

12899

18030

24973

23884

21852

19249

11326

6573

3852

2471

0

5000

10000

15000

20000

25000

30000


Input Amount

Ne

t S

ign

al In

ten

sit

y (

MF

I)

200 amol


81


0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000



Ne

t S

ign

al in

ten

sit

y (

MF

I)

82

miR-133a

000

020

040

060

080

100

120


Total RNA Samples

Rela

tive C

han

ge t

o H

eart

FlexmiR v2

qRT-PCR

Tissue

Re

lati

ve

Ch

an

ge


LNP-miRNA

qRT-PCR

83

miRNA

Re

lati

ve

Ch

an

ge



0

5

10

15

20

25


miRNA

Rela

tive C

han

ge

FlexmiR v2

qRT-PCR

LNP-miRNA

qPCR

84



0

5000

10000

15000

20000

25000

30000

hsa-m

iR-1

07

hsa-m

iR-1

47

hsa-m

iR-1

81a

hsa-m

iR-1

92

hsa-m

iR-1

9b

hsa-m

iR-2

20a

hsa-m

iR-2

1hsa-m

iR-2

18

hsa-le

t-7c

hsa-m

iR-1

00

hsa-m

iR-1

25b

hsa-m

iR-

hsa-m

iR-2

05

hsa-m

iR-2

21

hsa-m

iR-2

00a

hsa-m

iR-2

00c

hsa-m

iR-2

06

hsa-m

iR-1

0a

hsa-m

iR-1

6hsa-m

iR-9

hsa-m

iR-4

24

hsa-m

iR-2

0a

hsa-m

iR-2

0b

hsa-le

t-7a

hsa-m

iR-9

5hsa-m

iR-1

36

hsa-m

iR-1

95

hsa-m

iR-2

10

hsa-m

iR-2

12

hsa-m

iR-

hsa-m

iR-1

7hsa-m

iR-1

22

hsa-m

iR-1

43

hsa-m

iR-1

45

hsa-m

ir-1

06a

hsa-m

iR-

hsa-m

iR-1

0b

hsa-m

iR-1

hsa-m

iR-2

23

hsa-m

iR-1

81b

hsa-le

t-155

hsa-le

t-7g

hsa-m

iR-7

ihsa-m

iR-9

6hsa-m

iR-1

37

hsa-m

iR-1

85

hsa-m

iR-1

38

hsa-m

iR-1

33a

hsa-m

iR-1

33b

hsa-m

iR-1

5a

hsa-m

iR-3

4a

hsa-m

iR-2

9a

hsa-m

iR-2

9b

hsa-m

iR-1

03

hsa-m

iR-2

22

hsa-m

iR-1

46a

hsa-m

iR-3

9cel-m

iR-3

9cel-m

iR-2

38

Regions

Ne

t S

ign

al in

ten

sit

y (

MF

I)

50ng

100ng

250ng

500ng








bull Flexible





Proteomic Assays



Nucleic Acids








or IgM





Multiplex Detection

Detection


IgG

1 2 3 4





















Y Y

Y

IgG1 IgG2

IgG3

IgG4 IgA



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Detection



Antigen

1 2 3








Y Y Y

Y

Y

Y Y

Y Y

Y Y

Y



Y


Detection

Multiplex Detection


















compounds





compounds



SAPE SAPE














SAPE SAPE


Detection

Multiplex Detection












543-550






GTPases 827 271-282














Single Plex Assays

To Luminex

Multiplex

Assays





A

5rsquo

3rsquo

G 5rsquo

3rsquo

T








xTAG


Detection

SAPE










xTAG primer

gDNA


Biotinylated primer

5rsquo 3rsquo

3rsquo

dATP dTTP dGTP dCTP

= Carbon Spacer











Detection

Sequence Detection

TAG


















Workflow

75



2 hours



30 minutes



30 minutes





30 minutes

76






0

500

1000

1500

2000

2500

3000

3500

4000


Sample

Sig

nal In

ten

sit

y (

MF

I)

let-7c

Let-7a

77


miR-107 vs miR-103

0

1000

2000

3000

4000

5000

6000


Sample

Sig

na

l In

ten

sit

y (

MF

I)

miR-103

miR-107









0

1000

2000

3000

4000

5000

6000

7000

8000

9000

10000

hsa-m

iR-1

07

hsa-m

iR-1

47

hsa-m

iR-1

81a

hsa-m

iR-1

92

hsa-m

iR-1

9b

hsa-m

iR-2

20a

hsa-m

iR-2

1hsa-m

iR-2

18

hsa-le

t-7c

hsa-m

iR-1

00

hsa-m

iR-1

25b

hsa-m

iR-

hsa-m

iR-2

05

hsa-m

iR-2

21

hsa-m

iR-2

00a

hsa-m

iR-2

00c

hsa-m

iR-2

06

hsa-m

iR-1

0a

hsa-m

iR-1

6

hsa-m

iR-9

hsa-m

iR-4

24

hsa-m

iR-2

0a

hsa-m

iR-2

0b

hsa-le

t-7a

hsa-m

iR-9

5hsa-m

iR-1

36

hsa-m

iR-1

95

hsa-m

iR-2

10

hsa-m

iR-2

12

hsa-m

iR-

hsa-m

iR-1

7

hsa-m

iR-1

22

hsa-m

iR-1

43

hsa-m

iR-1

45

hsa-m

iR-1

06a

hsa-m

iR-

hsa-m

iR-1

0b

hsa-m

iR-1

hsa-m

iR-2

23

hsa-m

iR-1

81b

hsa-m

iR-1

55

hsa-le

t-7g

hsa-le

t-7i

hsa-m

iR-9

6

hsa-m

iR-1

37

hsa-m

iR-1

85

hsa-m

iR-1

38

hsa-m

iR-1

98

hsa-m

iR-1

33a

hsa-m

iR-1

33b

hsa-m

iR-1

5a

hsa-m

iR-3

4a

hsa-m

iR-2

9a

hsa-m

iR-2

9b

hsa-m

iR-1

03

hsa-m

iR-2

22

hsa-m

iR-1

46a

cel-m

iR-3

9cel-m

iR-5

4

cel-m

iR-2

38

Regions

Ne

t S

ign

al In

ten

sit

y (

MF

I)

106a

17


Total RNA Samples

53-Plex Assay

miR-9 miR-122 miR-1

0

5000

10000

15000

20000

25000

Samples

Ne

t S

ign

al In

ten

sit

y (

MF

I)

miR-9

miR-122

miR-1






289704

12431797

4050

7044

12899

18030

24973

23884

21852

19249

11326

6573

3852

2471

0

5000

10000

15000

20000

25000

30000


Input Amount

Ne

t S

ign

al In

ten

sit

y (

MF

I)

200 amol


81


0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000



Ne

t S

ign

al in

ten

sit

y (

MF

I)

82

miR-133a

000

020

040

060

080

100

120


Total RNA Samples

Rela

tive C

han

ge t

o H

eart

FlexmiR v2

qRT-PCR

Tissue

Re

lati

ve

Ch

an

ge


LNP-miRNA

qRT-PCR

83

miRNA

Re

lati

ve

Ch

an

ge



0

5

10

15

20

25


miRNA

Rela

tive C

han

ge

FlexmiR v2

qRT-PCR

LNP-miRNA

qPCR

84



0

5000

10000

15000

20000

25000

30000

hsa-m

iR-1

07

hsa-m

iR-1

47

hsa-m

iR-1

81a

hsa-m

iR-1

92

hsa-m

iR-1

9b

hsa-m

iR-2

20a

hsa-m

iR-2

1hsa-m

iR-2

18

hsa-le

t-7c

hsa-m

iR-1

00

hsa-m

iR-1

25b

hsa-m

iR-

hsa-m

iR-2

05

hsa-m

iR-2

21

hsa-m

iR-2

00a

hsa-m

iR-2

00c

hsa-m

iR-2

06

hsa-m

iR-1

0a

hsa-m

iR-1

6hsa-m

iR-9

hsa-m

iR-4

24

hsa-m

iR-2

0a

hsa-m

iR-2

0b

hsa-le

t-7a

hsa-m

iR-9

5hsa-m

iR-1

36

hsa-m

iR-1

95

hsa-m

iR-2

10

hsa-m

iR-2

12

hsa-m

iR-

hsa-m

iR-1

7hsa-m

iR-1

22

hsa-m

iR-1

43

hsa-m

iR-1

45

hsa-m

ir-1

06a

hsa-m

iR-

hsa-m

iR-1

0b

hsa-m

iR-1

hsa-m

iR-2

23

hsa-m

iR-1

81b

hsa-le

t-155

hsa-le

t-7g

hsa-m

iR-7

ihsa-m

iR-9

6hsa-m

iR-1

37

hsa-m

iR-1

85

hsa-m

iR-1

38

hsa-m

iR-1

33a

hsa-m

iR-1

33b

hsa-m

iR-1

5a

hsa-m

iR-3

4a

hsa-m

iR-2

9a

hsa-m

iR-2

9b

hsa-m

iR-1

03

hsa-m

iR-2

22

hsa-m

iR-1

46a

hsa-m

iR-3

9cel-m

iR-3

9cel-m

iR-2

38

Regions

Ne

t S

ign

al in

ten

sit

y (

MF

I)

50ng

100ng

250ng

500ng








bull Flexible





Proteomic Assays



Nucleic Acids








or IgM





Multiplex Detection

Detection


IgG

1 2 3 4





















Y Y

Y

IgG1 IgG2

IgG3

IgG4 IgA



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Detection



Antigen

1 2 3








Y Y Y

Y

Y

Y Y

Y Y

Y Y

Y



Y


Detection

Multiplex Detection


















compounds





compounds



SAPE SAPE














SAPE SAPE


Detection

Multiplex Detection












543-550






GTPases 827 271-282














Single Plex Assays

To Luminex

Multiplex

Assays









xTAG


Detection

SAPE










xTAG primer

gDNA


Biotinylated primer

5rsquo 3rsquo

3rsquo

dATP dTTP dGTP dCTP

= Carbon Spacer











Detection

Sequence Detection

TAG


















Workflow

75



2 hours



30 minutes



30 minutes





30 minutes

76






0

500

1000

1500

2000

2500

3000

3500

4000


Sample

Sig

nal In

ten

sit

y (

MF

I)

let-7c

Let-7a

77


miR-107 vs miR-103

0

1000

2000

3000

4000

5000

6000


Sample

Sig

na

l In

ten

sit

y (

MF

I)

miR-103

miR-107









0

1000

2000

3000

4000

5000

6000

7000

8000

9000

10000

hsa-m

iR-1

07

hsa-m

iR-1

47

hsa-m

iR-1

81a

hsa-m

iR-1

92

hsa-m

iR-1

9b

hsa-m

iR-2

20a

hsa-m

iR-2

1hsa-m

iR-2

18

hsa-le

t-7c

hsa-m

iR-1

00

hsa-m

iR-1

25b

hsa-m

iR-

hsa-m

iR-2

05

hsa-m

iR-2

21

hsa-m

iR-2

00a

hsa-m

iR-2

00c

hsa-m

iR-2

06

hsa-m

iR-1

0a

hsa-m

iR-1

6

hsa-m

iR-9

hsa-m

iR-4

24

hsa-m

iR-2

0a

hsa-m

iR-2

0b

hsa-le

t-7a

hsa-m

iR-9

5hsa-m

iR-1

36

hsa-m

iR-1

95

hsa-m

iR-2

10

hsa-m

iR-2

12

hsa-m

iR-

hsa-m

iR-1

7

hsa-m

iR-1

22

hsa-m

iR-1

43

hsa-m

iR-1

45

hsa-m

iR-1

06a

hsa-m

iR-

hsa-m

iR-1

0b

hsa-m

iR-1

hsa-m

iR-2

23

hsa-m

iR-1

81b

hsa-m

iR-1

55

hsa-le

t-7g

hsa-le

t-7i

hsa-m

iR-9

6

hsa-m

iR-1

37

hsa-m

iR-1

85

hsa-m

iR-1

38

hsa-m

iR-1

98

hsa-m

iR-1

33a

hsa-m

iR-1

33b

hsa-m

iR-1

5a

hsa-m

iR-3

4a

hsa-m

iR-2

9a

hsa-m

iR-2

9b

hsa-m

iR-1

03

hsa-m

iR-2

22

hsa-m

iR-1

46a

cel-m

iR-3

9cel-m

iR-5

4

cel-m

iR-2

38

Regions

Ne

t S

ign

al In

ten

sit

y (

MF

I)

106a

17


Total RNA Samples

53-Plex Assay

miR-9 miR-122 miR-1

0

5000

10000

15000

20000

25000

Samples

Ne

t S

ign

al In

ten

sit

y (

MF

I)

miR-9

miR-122

miR-1






289704

12431797

4050

7044

12899

18030

24973

23884

21852

19249

11326

6573

3852

2471

0

5000

10000

15000

20000

25000

30000


Input Amount

Ne

t S

ign

al In

ten

sit

y (

MF

I)

200 amol


81


0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000



Ne

t S

ign

al in

ten

sit

y (

MF

I)

82

miR-133a

000

020

040

060

080

100

120


Total RNA Samples

Rela

tive C

han

ge t

o H

eart

FlexmiR v2

qRT-PCR

Tissue

Re

lati

ve

Ch

an

ge


LNP-miRNA

qRT-PCR

83

miRNA

Re

lati

ve

Ch

an

ge



0

5

10

15

20

25


miRNA

Rela

tive C

han

ge

FlexmiR v2

qRT-PCR

LNP-miRNA

qPCR

84



0

5000

10000

15000

20000

25000

30000

hsa-m

iR-1

07

hsa-m

iR-1

47

hsa-m

iR-1

81a

hsa-m

iR-1

92

hsa-m

iR-1

9b

hsa-m

iR-2

20a

hsa-m

iR-2

1hsa-m

iR-2

18

hsa-le

t-7c

hsa-m

iR-1

00

hsa-m

iR-1

25b

hsa-m

iR-

hsa-m

iR-2

05

hsa-m

iR-2

21

hsa-m

iR-2

00a

hsa-m

iR-2

00c

hsa-m

iR-2

06

hsa-m

iR-1

0a

hsa-m

iR-1

6hsa-m

iR-9

hsa-m

iR-4

24

hsa-m

iR-2

0a

hsa-m

iR-2

0b

hsa-le

t-7a

hsa-m

iR-9

5hsa-m

iR-1

36

hsa-m

iR-1

95

hsa-m

iR-2

10

hsa-m

iR-2

12

hsa-m

iR-

hsa-m

iR-1

7hsa-m

iR-1

22

hsa-m

iR-1

43

hsa-m

iR-1

45

hsa-m

ir-1

06a

hsa-m

iR-

hsa-m

iR-1

0b

hsa-m

iR-1

hsa-m

iR-2

23

hsa-m

iR-1

81b

hsa-le

t-155

hsa-le

t-7g

hsa-m

iR-7

ihsa-m

iR-9

6hsa-m

iR-1

37

hsa-m

iR-1

85

hsa-m

iR-1

38

hsa-m

iR-1

33a

hsa-m

iR-1

33b

hsa-m

iR-1

5a

hsa-m

iR-3

4a

hsa-m

iR-2

9a

hsa-m

iR-2

9b

hsa-m

iR-1

03

hsa-m

iR-2

22

hsa-m

iR-1

46a

hsa-m

iR-3

9cel-m

iR-3

9cel-m

iR-2

38

Regions

Ne

t S

ign

al in

ten

sit

y (

MF

I)

50ng

100ng

250ng

500ng








bull Flexible





Proteomic Assays



Nucleic Acids








or IgM





Multiplex Detection

Detection


IgG

1 2 3 4





















Y Y

Y

IgG1 IgG2

IgG3

IgG4 IgA



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Detection



Antigen

1 2 3








Y Y Y

Y

Y

Y Y

Y Y

Y Y

Y



Y


Detection

Multiplex Detection


















compounds





compounds



SAPE SAPE














SAPE SAPE


Detection

Multiplex Detection












543-550






GTPases 827 271-282














Single Plex Assays

To Luminex

Multiplex

Assays







xTAG


Detection

SAPE










xTAG primer

gDNA


Biotinylated primer

5rsquo 3rsquo

3rsquo

dATP dTTP dGTP dCTP

= Carbon Spacer











Detection

Sequence Detection

TAG


















Workflow

75



2 hours



30 minutes



30 minutes





30 minutes

76






0

500

1000

1500

2000

2500

3000

3500

4000


Sample

Sig

nal In

ten

sit

y (

MF

I)

let-7c

Let-7a

77


miR-107 vs miR-103

0

1000

2000

3000

4000

5000

6000


Sample

Sig

na

l In

ten

sit

y (

MF

I)

miR-103

miR-107









0

1000

2000

3000

4000

5000

6000

7000

8000

9000

10000

hsa-m

iR-1

07

hsa-m

iR-1

47

hsa-m

iR-1

81a

hsa-m

iR-1

92

hsa-m

iR-1

9b

hsa-m

iR-2

20a

hsa-m

iR-2

1hsa-m

iR-2

18

hsa-le

t-7c

hsa-m

iR-1

00

hsa-m

iR-1

25b

hsa-m

iR-

hsa-m

iR-2

05

hsa-m

iR-2

21

hsa-m

iR-2

00a

hsa-m

iR-2

00c

hsa-m

iR-2

06

hsa-m

iR-1

0a

hsa-m

iR-1

6

hsa-m

iR-9

hsa-m

iR-4

24

hsa-m

iR-2

0a

hsa-m

iR-2

0b

hsa-le

t-7a

hsa-m

iR-9

5hsa-m

iR-1

36

hsa-m

iR-1

95

hsa-m

iR-2

10

hsa-m

iR-2

12

hsa-m

iR-

hsa-m

iR-1

7

hsa-m

iR-1

22

hsa-m

iR-1

43

hsa-m

iR-1

45

hsa-m

iR-1

06a

hsa-m

iR-

hsa-m

iR-1

0b

hsa-m

iR-1

hsa-m

iR-2

23

hsa-m

iR-1

81b

hsa-m

iR-1

55

hsa-le

t-7g

hsa-le

t-7i

hsa-m

iR-9

6

hsa-m

iR-1

37

hsa-m

iR-1

85

hsa-m

iR-1

38

hsa-m

iR-1

98

hsa-m

iR-1

33a

hsa-m

iR-1

33b

hsa-m

iR-1

5a

hsa-m

iR-3

4a

hsa-m

iR-2

9a

hsa-m

iR-2

9b

hsa-m

iR-1

03

hsa-m

iR-2

22

hsa-m

iR-1

46a

cel-m

iR-3

9cel-m

iR-5

4

cel-m

iR-2

38

Regions

Ne

t S

ign

al In

ten

sit

y (

MF

I)

106a

17


Total RNA Samples

53-Plex Assay

miR-9 miR-122 miR-1

0

5000

10000

15000

20000

25000

Samples

Ne

t S

ign

al In

ten

sit

y (

MF

I)

miR-9

miR-122

miR-1






289704

12431797

4050

7044

12899

18030

24973

23884

21852

19249

11326

6573

3852

2471

0

5000

10000

15000

20000

25000

30000


Input Amount

Ne

t S

ign

al In

ten

sit

y (

MF

I)

200 amol


81


0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000



Ne

t S

ign

al in

ten

sit

y (

MF

I)

82

miR-133a

000

020

040

060

080

100

120


Total RNA Samples

Rela

tive C

han

ge t

o H

eart

FlexmiR v2

qRT-PCR

Tissue

Re

lati

ve

Ch

an

ge


LNP-miRNA

qRT-PCR

83

miRNA

Re

lati

ve

Ch

an

ge



0

5

10

15

20

25


miRNA

Rela

tive C

han

ge

FlexmiR v2

qRT-PCR

LNP-miRNA

qPCR

84



0

5000

10000

15000

20000

25000

30000

hsa-m

iR-1

07

hsa-m

iR-1

47

hsa-m

iR-1

81a

hsa-m

iR-1

92

hsa-m

iR-1

9b

hsa-m

iR-2

20a

hsa-m

iR-2

1hsa-m

iR-2

18

hsa-le

t-7c

hsa-m

iR-1

00

hsa-m

iR-1

25b

hsa-m

iR-

hsa-m

iR-2

05

hsa-m

iR-2

21

hsa-m

iR-2

00a

hsa-m

iR-2

00c

hsa-m

iR-2

06

hsa-m

iR-1

0a

hsa-m

iR-1

6hsa-m

iR-9

hsa-m

iR-4

24

hsa-m

iR-2

0a

hsa-m

iR-2

0b

hsa-le

t-7a

hsa-m

iR-9

5hsa-m

iR-1

36

hsa-m

iR-1

95

hsa-m

iR-2

10

hsa-m

iR-2

12

hsa-m

iR-

hsa-m

iR-1

7hsa-m

iR-1

22

hsa-m

iR-1

43

hsa-m

iR-1

45

hsa-m

ir-1

06a

hsa-m

iR-

hsa-m

iR-1

0b

hsa-m

iR-1

hsa-m

iR-2

23

hsa-m

iR-1

81b

hsa-le

t-155

hsa-le

t-7g

hsa-m

iR-7

ihsa-m

iR-9

6hsa-m

iR-1

37

hsa-m

iR-1

85

hsa-m

iR-1

38

hsa-m

iR-1

33a

hsa-m

iR-1

33b

hsa-m

iR-1

5a

hsa-m

iR-3

4a

hsa-m

iR-2

9a

hsa-m

iR-2

9b

hsa-m

iR-1

03

hsa-m

iR-2

22

hsa-m

iR-1

46a

hsa-m

iR-3

9cel-m

iR-3

9cel-m

iR-2

38

Regions

Ne

t S

ign

al in

ten

sit

y (

MF

I)

50ng

100ng

250ng

500ng








bull Flexible





Proteomic Assays



Nucleic Acids








or IgM





Multiplex Detection

Detection


IgG

1 2 3 4





















Y Y

Y

IgG1 IgG2

IgG3

IgG4 IgA



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Detection



Antigen

1 2 3








Y Y Y

Y

Y

Y Y

Y Y

Y Y

Y



Y


Detection

Multiplex Detection


















compounds





compounds



SAPE SAPE














SAPE SAPE


Detection

Multiplex Detection












543-550






GTPases 827 271-282














Single Plex Assays

To Luminex

Multiplex

Assays











xTAG primer

gDNA


Biotinylated primer

5rsquo 3rsquo

3rsquo

dATP dTTP dGTP dCTP

= Carbon Spacer











Detection

Sequence Detection

TAG


















Workflow

75



2 hours



30 minutes



30 minutes





30 minutes

76






0

500

1000

1500

2000

2500

3000

3500

4000


Sample

Sig

nal In

ten

sit

y (

MF

I)

let-7c

Let-7a

77


miR-107 vs miR-103

0

1000

2000

3000

4000

5000

6000


Sample

Sig

na

l In

ten

sit

y (

MF

I)

miR-103

miR-107









0

1000

2000

3000

4000

5000

6000

7000

8000

9000

10000

hsa-m

iR-1

07

hsa-m

iR-1

47

hsa-m

iR-1

81a

hsa-m

iR-1

92

hsa-m

iR-1

9b

hsa-m

iR-2

20a

hsa-m

iR-2

1hsa-m

iR-2

18

hsa-le

t-7c

hsa-m

iR-1

00

hsa-m

iR-1

25b

hsa-m

iR-

hsa-m

iR-2

05

hsa-m

iR-2

21

hsa-m

iR-2

00a

hsa-m

iR-2

00c

hsa-m

iR-2

06

hsa-m

iR-1

0a

hsa-m

iR-1

6

hsa-m

iR-9

hsa-m

iR-4

24

hsa-m

iR-2

0a

hsa-m

iR-2

0b

hsa-le

t-7a

hsa-m

iR-9

5hsa-m

iR-1

36

hsa-m

iR-1

95

hsa-m

iR-2

10

hsa-m

iR-2

12

hsa-m

iR-

hsa-m

iR-1

7

hsa-m

iR-1

22

hsa-m

iR-1

43

hsa-m

iR-1

45

hsa-m

iR-1

06a

hsa-m

iR-

hsa-m

iR-1

0b

hsa-m

iR-1

hsa-m

iR-2

23

hsa-m

iR-1

81b

hsa-m

iR-1

55

hsa-le

t-7g

hsa-le

t-7i

hsa-m

iR-9

6

hsa-m

iR-1

37

hsa-m

iR-1

85

hsa-m

iR-1

38

hsa-m

iR-1

98

hsa-m

iR-1

33a

hsa-m

iR-1

33b

hsa-m

iR-1

5a

hsa-m

iR-3

4a

hsa-m

iR-2

9a

hsa-m

iR-2

9b

hsa-m

iR-1

03

hsa-m

iR-2

22

hsa-m

iR-1

46a

cel-m

iR-3

9cel-m

iR-5

4

cel-m

iR-2

38

Regions

Ne

t S

ign

al In

ten

sit

y (

MF

I)

106a

17


Total RNA Samples

53-Plex Assay

miR-9 miR-122 miR-1

0

5000

10000

15000

20000

25000

Samples

Ne

t S

ign

al In

ten

sit

y (

MF

I)

miR-9

miR-122

miR-1






289704

12431797

4050

7044

12899

18030

24973

23884

21852

19249

11326

6573

3852

2471

0

5000

10000

15000

20000

25000

30000


Input Amount

Ne

t S

ign

al In

ten

sit

y (

MF

I)

200 amol


81


0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000



Ne

t S

ign

al in

ten

sit

y (

MF

I)

82

miR-133a

000

020

040

060

080

100

120


Total RNA Samples

Rela

tive C

han

ge t

o H

eart

FlexmiR v2

qRT-PCR

Tissue

Re

lati

ve

Ch

an

ge


LNP-miRNA

qRT-PCR

83

miRNA

Re

lati

ve

Ch

an

ge



0

5

10

15

20

25


miRNA

Rela

tive C

han

ge

FlexmiR v2

qRT-PCR

LNP-miRNA

qPCR

84



0

5000

10000

15000

20000

25000

30000

hsa-m

iR-1

07

hsa-m

iR-1

47

hsa-m

iR-1

81a

hsa-m

iR-1

92

hsa-m

iR-1

9b

hsa-m

iR-2

20a

hsa-m

iR-2

1hsa-m

iR-2

18

hsa-le

t-7c

hsa-m

iR-1

00

hsa-m

iR-1

25b

hsa-m

iR-

hsa-m

iR-2

05

hsa-m

iR-2

21

hsa-m

iR-2

00a

hsa-m

iR-2

00c

hsa-m

iR-2

06

hsa-m

iR-1

0a

hsa-m

iR-1

6hsa-m

iR-9

hsa-m

iR-4

24

hsa-m

iR-2

0a

hsa-m

iR-2

0b

hsa-le

t-7a

hsa-m

iR-9

5hsa-m

iR-1

36

hsa-m

iR-1

95

hsa-m

iR-2

10

hsa-m

iR-2

12

hsa-m

iR-

hsa-m

iR-1

7hsa-m

iR-1

22

hsa-m

iR-1

43

hsa-m

iR-1

45

hsa-m

ir-1

06a

hsa-m

iR-

hsa-m

iR-1

0b

hsa-m

iR-1

hsa-m

iR-2

23

hsa-m

iR-1

81b

hsa-le

t-155

hsa-le

t-7g

hsa-m

iR-7

ihsa-m

iR-9

6hsa-m

iR-1

37

hsa-m

iR-1

85

hsa-m

iR-1

38

hsa-m

iR-1

33a

hsa-m

iR-1

33b

hsa-m

iR-1

5a

hsa-m

iR-3

4a

hsa-m

iR-2

9a

hsa-m

iR-2

9b

hsa-m

iR-1

03

hsa-m

iR-2

22

hsa-m

iR-1

46a

hsa-m

iR-3

9cel-m

iR-3

9cel-m

iR-2

38

Regions

Ne

t S

ign

al in

ten

sit

y (

MF

I)

50ng

100ng

250ng

500ng








bull Flexible





Proteomic Assays



Nucleic Acids








or IgM





Multiplex Detection

Detection


IgG

1 2 3 4





















Y Y

Y

IgG1 IgG2

IgG3

IgG4 IgA



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Detection



Antigen

1 2 3








Y Y Y

Y

Y

Y Y

Y Y

Y Y

Y



Y


Detection

Multiplex Detection


















compounds





compounds



SAPE SAPE














SAPE SAPE


Detection

Multiplex Detection












543-550






GTPases 827 271-282














Single Plex Assays

To Luminex

Multiplex

Assays








xTAG primer

gDNA


Biotinylated primer

5rsquo 3rsquo

3rsquo

dATP dTTP dGTP dCTP

= Carbon Spacer











Detection

Sequence Detection

TAG


















Workflow

75



2 hours



30 minutes



30 minutes





30 minutes

76






0

500

1000

1500

2000

2500

3000

3500

4000


Sample

Sig

nal In

ten

sit

y (

MF

I)

let-7c

Let-7a

77


miR-107 vs miR-103

0

1000

2000

3000

4000

5000

6000


Sample

Sig

na

l In

ten

sit

y (

MF

I)

miR-103

miR-107









0

1000

2000

3000

4000

5000

6000

7000

8000

9000

10000

hsa-m

iR-1

07

hsa-m

iR-1

47

hsa-m

iR-1

81a

hsa-m

iR-1

92

hsa-m

iR-1

9b

hsa-m

iR-2

20a

hsa-m

iR-2

1hsa-m

iR-2

18

hsa-le

t-7c

hsa-m

iR-1

00

hsa-m

iR-1

25b

hsa-m

iR-

hsa-m

iR-2

05

hsa-m

iR-2

21

hsa-m

iR-2

00a

hsa-m

iR-2

00c

hsa-m

iR-2

06

hsa-m

iR-1

0a

hsa-m

iR-1

6

hsa-m

iR-9

hsa-m

iR-4

24

hsa-m

iR-2

0a

hsa-m

iR-2

0b

hsa-le

t-7a

hsa-m

iR-9

5hsa-m

iR-1

36

hsa-m

iR-1

95

hsa-m

iR-2

10

hsa-m

iR-2

12

hsa-m

iR-

hsa-m

iR-1

7

hsa-m

iR-1

22

hsa-m

iR-1

43

hsa-m

iR-1

45

hsa-m

iR-1

06a

hsa-m

iR-

hsa-m

iR-1

0b

hsa-m

iR-1

hsa-m

iR-2

23

hsa-m

iR-1

81b

hsa-m

iR-1

55

hsa-le

t-7g

hsa-le

t-7i

hsa-m

iR-9

6

hsa-m

iR-1

37

hsa-m

iR-1

85

hsa-m

iR-1

38

hsa-m

iR-1

98

hsa-m

iR-1

33a

hsa-m

iR-1

33b

hsa-m

iR-1

5a

hsa-m

iR-3

4a

hsa-m

iR-2

9a

hsa-m

iR-2

9b

hsa-m

iR-1

03

hsa-m

iR-2

22

hsa-m

iR-1

46a

cel-m

iR-3

9cel-m

iR-5

4

cel-m

iR-2

38

Regions

Ne

t S

ign

al In

ten

sit

y (

MF

I)

106a

17


Total RNA Samples

53-Plex Assay

miR-9 miR-122 miR-1

0

5000

10000

15000

20000

25000

Samples

Ne

t S

ign

al In

ten

sit

y (

MF

I)

miR-9

miR-122

miR-1






289704

12431797

4050

7044

12899

18030

24973

23884

21852

19249

11326

6573

3852

2471

0

5000

10000

15000

20000

25000

30000


Input Amount

Ne

t S

ign

al In

ten

sit

y (

MF

I)

200 amol


81


0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000



Ne

t S

ign

al in

ten

sit

y (

MF

I)

82

miR-133a

000

020

040

060

080

100

120


Total RNA Samples

Rela

tive C

han

ge t

o H

eart

FlexmiR v2

qRT-PCR

Tissue

Re

lati

ve

Ch

an

ge


LNP-miRNA

qRT-PCR

83

miRNA

Re

lati

ve

Ch

an

ge



0

5

10

15

20

25


miRNA

Rela

tive C

han

ge

FlexmiR v2

qRT-PCR

LNP-miRNA

qPCR

84



0

5000

10000

15000

20000

25000

30000

hsa-m

iR-1

07

hsa-m

iR-1

47

hsa-m

iR-1

81a

hsa-m

iR-1

92

hsa-m

iR-1

9b

hsa-m

iR-2

20a

hsa-m

iR-2

1hsa-m

iR-2

18

hsa-le

t-7c

hsa-m

iR-1

00

hsa-m

iR-1

25b

hsa-m

iR-

hsa-m

iR-2

05

hsa-m

iR-2

21

hsa-m

iR-2

00a

hsa-m

iR-2

00c

hsa-m

iR-2

06

hsa-m

iR-1

0a

hsa-m

iR-1

6hsa-m

iR-9

hsa-m

iR-4

24

hsa-m

iR-2

0a

hsa-m

iR-2

0b

hsa-le

t-7a

hsa-m

iR-9

5hsa-m

iR-1

36

hsa-m

iR-1

95

hsa-m

iR-2

10

hsa-m

iR-2

12

hsa-m

iR-

hsa-m

iR-1

7hsa-m

iR-1

22

hsa-m

iR-1

43

hsa-m

iR-1

45

hsa-m

ir-1

06a

hsa-m

iR-

hsa-m

iR-1

0b

hsa-m

iR-1

hsa-m

iR-2

23

hsa-m

iR-1

81b

hsa-le

t-155

hsa-le

t-7g

hsa-m

iR-7

ihsa-m

iR-9

6hsa-m

iR-1

37

hsa-m

iR-1

85

hsa-m

iR-1

38

hsa-m

iR-1

33a

hsa-m

iR-1

33b

hsa-m

iR-1

5a

hsa-m

iR-3

4a

hsa-m

iR-2

9a

hsa-m

iR-2

9b

hsa-m

iR-1

03

hsa-m

iR-2

22

hsa-m

iR-1

46a

hsa-m

iR-3

9cel-m

iR-3

9cel-m

iR-2

38

Regions

Ne

t S

ign

al in

ten

sit

y (

MF

I)

50ng

100ng

250ng

500ng








bull Flexible





Proteomic Assays



Nucleic Acids








or IgM





Multiplex Detection

Detection


IgG

1 2 3 4





















Y Y

Y

IgG1 IgG2

IgG3

IgG4 IgA



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Detection



Antigen

1 2 3








Y Y Y

Y

Y

Y Y

Y Y

Y Y

Y



Y


Detection

Multiplex Detection


















compounds





compounds



SAPE SAPE














SAPE SAPE


Detection

Multiplex Detection












543-550






GTPases 827 271-282














Single Plex Assays

To Luminex

Multiplex

Assays







xTAG primer

gDNA


Biotinylated primer

5rsquo 3rsquo

3rsquo

dATP dTTP dGTP dCTP

= Carbon Spacer











Detection

Sequence Detection

TAG


















Workflow

75



2 hours



30 minutes



30 minutes





30 minutes

76






0

500

1000

1500

2000

2500

3000

3500

4000


Sample

Sig

nal In

ten

sit

y (

MF

I)

let-7c

Let-7a

77


miR-107 vs miR-103

0

1000

2000

3000

4000

5000

6000


Sample

Sig

na

l In

ten

sit

y (

MF

I)

miR-103

miR-107









0

1000

2000

3000

4000

5000

6000

7000

8000

9000

10000

hsa-m

iR-1

07

hsa-m

iR-1

47

hsa-m

iR-1

81a

hsa-m

iR-1

92

hsa-m

iR-1

9b

hsa-m

iR-2

20a

hsa-m

iR-2

1hsa-m

iR-2

18

hsa-le

t-7c

hsa-m

iR-1

00

hsa-m

iR-1

25b

hsa-m

iR-

hsa-m

iR-2

05

hsa-m

iR-2

21

hsa-m

iR-2

00a

hsa-m

iR-2

00c

hsa-m

iR-2

06

hsa-m

iR-1

0a

hsa-m

iR-1

6

hsa-m

iR-9

hsa-m

iR-4

24

hsa-m

iR-2

0a

hsa-m

iR-2

0b

hsa-le

t-7a

hsa-m

iR-9

5hsa-m

iR-1

36

hsa-m

iR-1

95

hsa-m

iR-2

10

hsa-m

iR-2

12

hsa-m

iR-

hsa-m

iR-1

7

hsa-m

iR-1

22

hsa-m

iR-1

43

hsa-m

iR-1

45

hsa-m

iR-1

06a

hsa-m

iR-

hsa-m

iR-1

0b

hsa-m

iR-1

hsa-m

iR-2

23

hsa-m

iR-1

81b

hsa-m

iR-1

55

hsa-le

t-7g

hsa-le

t-7i

hsa-m

iR-9

6

hsa-m

iR-1

37

hsa-m

iR-1

85

hsa-m

iR-1

38

hsa-m

iR-1

98

hsa-m

iR-1

33a

hsa-m

iR-1

33b

hsa-m

iR-1

5a

hsa-m

iR-3

4a

hsa-m

iR-2

9a

hsa-m

iR-2

9b

hsa-m

iR-1

03

hsa-m

iR-2

22

hsa-m

iR-1

46a

cel-m

iR-3

9cel-m

iR-5

4

cel-m

iR-2

38

Regions

Ne

t S

ign

al In

ten

sit

y (

MF

I)

106a

17


Total RNA Samples

53-Plex Assay

miR-9 miR-122 miR-1

0

5000

10000

15000

20000

25000

Samples

Ne

t S

ign

al In

ten

sit

y (

MF

I)

miR-9

miR-122

miR-1






289704

12431797

4050

7044

12899

18030

24973

23884

21852

19249

11326

6573

3852

2471

0

5000

10000

15000

20000

25000

30000


Input Amount

Ne

t S

ign

al In

ten

sit

y (

MF

I)

200 amol


81


0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000



Ne

t S

ign

al in

ten

sit

y (

MF

I)

82

miR-133a

000

020

040

060

080

100

120


Total RNA Samples

Rela

tive C

han

ge t

o H

eart

FlexmiR v2

qRT-PCR

Tissue

Re

lati

ve

Ch

an

ge


LNP-miRNA

qRT-PCR

83

miRNA

Re

lati

ve

Ch

an

ge



0

5

10

15

20

25


miRNA

Rela

tive C

han

ge

FlexmiR v2

qRT-PCR

LNP-miRNA

qPCR

84



0

5000

10000

15000

20000

25000

30000

hsa-m

iR-1

07

hsa-m

iR-1

47

hsa-m

iR-1

81a

hsa-m

iR-1

92

hsa-m

iR-1

9b

hsa-m

iR-2

20a

hsa-m

iR-2

1hsa-m

iR-2

18

hsa-le

t-7c

hsa-m

iR-1

00

hsa-m

iR-1

25b

hsa-m

iR-

hsa-m

iR-2

05

hsa-m

iR-2

21

hsa-m

iR-2

00a

hsa-m

iR-2

00c

hsa-m

iR-2

06

hsa-m

iR-1

0a

hsa-m

iR-1

6hsa-m

iR-9

hsa-m

iR-4

24

hsa-m

iR-2

0a

hsa-m

iR-2

0b

hsa-le

t-7a

hsa-m

iR-9

5hsa-m

iR-1

36

hsa-m

iR-1

95

hsa-m

iR-2

10

hsa-m

iR-2

12

hsa-m

iR-

hsa-m

iR-1

7hsa-m

iR-1

22

hsa-m

iR-1

43

hsa-m

iR-1

45

hsa-m

ir-1

06a

hsa-m

iR-

hsa-m

iR-1

0b

hsa-m

iR-1

hsa-m

iR-2

23

hsa-m

iR-1

81b

hsa-le

t-155

hsa-le

t-7g

hsa-m

iR-7

ihsa-m

iR-9

6hsa-m

iR-1

37

hsa-m

iR-1

85

hsa-m

iR-1

38

hsa-m

iR-1

33a

hsa-m

iR-1

33b

hsa-m

iR-1

5a

hsa-m

iR-3

4a

hsa-m

iR-2

9a

hsa-m

iR-2

9b

hsa-m

iR-1

03

hsa-m

iR-2

22

hsa-m

iR-1

46a

hsa-m

iR-3

9cel-m

iR-3

9cel-m

iR-2

38

Regions

Ne

t S

ign

al in

ten

sit

y (

MF

I)

50ng

100ng

250ng

500ng








bull Flexible





Proteomic Assays



Nucleic Acids








or IgM





Multiplex Detection

Detection


IgG

1 2 3 4





















Y Y

Y

IgG1 IgG2

IgG3

IgG4 IgA



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Detection



Antigen

1 2 3








Y Y Y

Y

Y

Y Y

Y Y

Y Y

Y



Y


Detection

Multiplex Detection


















compounds





compounds



SAPE SAPE














SAPE SAPE


Detection

Multiplex Detection












543-550






GTPases 827 271-282














Single Plex Assays

To Luminex

Multiplex

Assays





xTAG primer

gDNA


Biotinylated primer

5rsquo 3rsquo

3rsquo

dATP dTTP dGTP dCTP

= Carbon Spacer











Detection

Sequence Detection

TAG


















Workflow

75



2 hours



30 minutes



30 minutes





30 minutes

76






0

500

1000

1500

2000

2500

3000

3500

4000


Sample

Sig

nal In

ten

sit

y (

MF

I)

let-7c

Let-7a

77


miR-107 vs miR-103

0

1000

2000

3000

4000

5000

6000


Sample

Sig

na

l In

ten

sit

y (

MF

I)

miR-103

miR-107









0

1000

2000

3000

4000

5000

6000

7000

8000

9000

10000

hsa-m

iR-1

07

hsa-m

iR-1

47

hsa-m

iR-1

81a

hsa-m

iR-1

92

hsa-m

iR-1

9b

hsa-m

iR-2

20a

hsa-m

iR-2

1hsa-m

iR-2

18

hsa-le

t-7c

hsa-m

iR-1

00

hsa-m

iR-1

25b

hsa-m

iR-

hsa-m

iR-2

05

hsa-m

iR-2

21

hsa-m

iR-2

00a

hsa-m

iR-2

00c

hsa-m

iR-2

06

hsa-m

iR-1

0a

hsa-m

iR-1

6

hsa-m

iR-9

hsa-m

iR-4

24

hsa-m

iR-2

0a

hsa-m

iR-2

0b

hsa-le

t-7a

hsa-m

iR-9

5hsa-m

iR-1

36

hsa-m

iR-1

95

hsa-m

iR-2

10

hsa-m

iR-2

12

hsa-m

iR-

hsa-m

iR-1

7

hsa-m

iR-1

22

hsa-m

iR-1

43

hsa-m

iR-1

45

hsa-m

iR-1

06a

hsa-m

iR-

hsa-m

iR-1

0b

hsa-m

iR-1

hsa-m

iR-2

23

hsa-m

iR-1

81b

hsa-m

iR-1

55

hsa-le

t-7g

hsa-le

t-7i

hsa-m

iR-9

6

hsa-m

iR-1

37

hsa-m

iR-1

85

hsa-m

iR-1

38

hsa-m

iR-1

98

hsa-m

iR-1

33a

hsa-m

iR-1

33b

hsa-m

iR-1

5a

hsa-m

iR-3

4a

hsa-m

iR-2

9a

hsa-m

iR-2

9b

hsa-m

iR-1

03

hsa-m

iR-2

22

hsa-m

iR-1

46a

cel-m

iR-3

9cel-m

iR-5

4

cel-m

iR-2

38

Regions

Ne

t S

ign

al In

ten

sit

y (

MF

I)

106a

17


Total RNA Samples

53-Plex Assay

miR-9 miR-122 miR-1

0

5000

10000

15000

20000

25000

Samples

Ne

t S

ign

al In

ten

sit

y (

MF

I)

miR-9

miR-122

miR-1






289704

12431797

4050

7044

12899

18030

24973

23884

21852

19249

11326

6573

3852

2471

0

5000

10000

15000

20000

25000

30000


Input Amount

Ne

t S

ign

al In

ten

sit

y (

MF

I)

200 amol


81


0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000



Ne

t S

ign

al in

ten

sit

y (

MF

I)

82

miR-133a

000

020

040

060

080

100

120


Total RNA Samples

Rela

tive C

han

ge t

o H

eart

FlexmiR v2

qRT-PCR

Tissue

Re

lati

ve

Ch

an

ge


LNP-miRNA

qRT-PCR

83

miRNA

Re

lati

ve

Ch

an

ge



0

5

10

15

20

25


miRNA

Rela

tive C

han

ge

FlexmiR v2

qRT-PCR

LNP-miRNA

qPCR

84



0

5000

10000

15000

20000

25000

30000

hsa-m

iR-1

07

hsa-m

iR-1

47

hsa-m

iR-1

81a

hsa-m

iR-1

92

hsa-m

iR-1

9b

hsa-m

iR-2

20a

hsa-m

iR-2

1hsa-m

iR-2

18

hsa-le

t-7c

hsa-m

iR-1

00

hsa-m

iR-1

25b

hsa-m

iR-

hsa-m

iR-2

05

hsa-m

iR-2

21

hsa-m

iR-2

00a

hsa-m

iR-2

00c

hsa-m

iR-2

06

hsa-m

iR-1

0a

hsa-m

iR-1

6hsa-m

iR-9

hsa-m

iR-4

24

hsa-m

iR-2

0a

hsa-m

iR-2

0b

hsa-le

t-7a

hsa-m

iR-9

5hsa-m

iR-1

36

hsa-m

iR-1

95

hsa-m

iR-2

10

hsa-m

iR-2

12

hsa-m

iR-

hsa-m

iR-1

7hsa-m

iR-1

22

hsa-m

iR-1

43

hsa-m

iR-1

45

hsa-m

ir-1

06a

hsa-m

iR-

hsa-m

iR-1

0b

hsa-m

iR-1

hsa-m

iR-2

23

hsa-m

iR-1

81b

hsa-le

t-155

hsa-le

t-7g

hsa-m

iR-7

ihsa-m

iR-9

6hsa-m

iR-1

37

hsa-m

iR-1

85

hsa-m

iR-1

38

hsa-m

iR-1

33a

hsa-m

iR-1

33b

hsa-m

iR-1

5a

hsa-m

iR-3

4a

hsa-m

iR-2

9a

hsa-m

iR-2

9b

hsa-m

iR-1

03

hsa-m

iR-2

22

hsa-m

iR-1

46a

hsa-m

iR-3

9cel-m

iR-3

9cel-m

iR-2

38

Regions

Ne

t S

ign

al in

ten

sit

y (

MF

I)

50ng

100ng

250ng

500ng








bull Flexible





Proteomic Assays



Nucleic Acids








or IgM





Multiplex Detection

Detection


IgG

1 2 3 4





















Y Y

Y

IgG1 IgG2

IgG3

IgG4 IgA



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Detection



Antigen

1 2 3








Y Y Y

Y

Y

Y Y

Y Y

Y Y

Y



Y


Detection

Multiplex Detection


















compounds





compounds



SAPE SAPE














SAPE SAPE


Detection

Multiplex Detection












543-550






GTPases 827 271-282














Single Plex Assays

To Luminex

Multiplex

Assays













Detection

Sequence Detection

TAG


















Workflow

75



2 hours



30 minutes



30 minutes





30 minutes

76






0

500

1000

1500

2000

2500

3000

3500

4000


Sample

Sig

nal In

ten

sit

y (

MF

I)

let-7c

Let-7a

77


miR-107 vs miR-103

0

1000

2000

3000

4000

5000

6000


Sample

Sig

na

l In

ten

sit

y (

MF

I)

miR-103

miR-107









0

1000

2000

3000

4000

5000

6000

7000

8000

9000

10000

hsa-m

iR-1

07

hsa-m

iR-1

47

hsa-m

iR-1

81a

hsa-m

iR-1

92

hsa-m

iR-1

9b

hsa-m

iR-2

20a

hsa-m

iR-2

1hsa-m

iR-2

18

hsa-le

t-7c

hsa-m

iR-1

00

hsa-m

iR-1

25b

hsa-m

iR-

hsa-m

iR-2

05

hsa-m

iR-2

21

hsa-m

iR-2

00a

hsa-m

iR-2

00c

hsa-m

iR-2

06

hsa-m

iR-1

0a

hsa-m

iR-1

6

hsa-m

iR-9

hsa-m

iR-4

24

hsa-m

iR-2

0a

hsa-m

iR-2

0b

hsa-le

t-7a

hsa-m

iR-9

5hsa-m

iR-1

36

hsa-m

iR-1

95

hsa-m

iR-2

10

hsa-m

iR-2

12

hsa-m

iR-

hsa-m

iR-1

7

hsa-m

iR-1

22

hsa-m

iR-1

43

hsa-m

iR-1

45

hsa-m

iR-1

06a

hsa-m

iR-

hsa-m

iR-1

0b

hsa-m

iR-1

hsa-m

iR-2

23

hsa-m

iR-1

81b

hsa-m

iR-1

55

hsa-le

t-7g

hsa-le

t-7i

hsa-m

iR-9

6

hsa-m

iR-1

37

hsa-m

iR-1

85

hsa-m

iR-1

38

hsa-m

iR-1

98

hsa-m

iR-1

33a

hsa-m

iR-1

33b

hsa-m

iR-1

5a

hsa-m

iR-3

4a

hsa-m

iR-2

9a

hsa-m

iR-2

9b

hsa-m

iR-1

03

hsa-m

iR-2

22

hsa-m

iR-1

46a

cel-m

iR-3

9cel-m

iR-5

4

cel-m

iR-2

38

Regions

Ne

t S

ign

al In

ten

sit

y (

MF

I)

106a

17


Total RNA Samples

53-Plex Assay

miR-9 miR-122 miR-1

0

5000

10000

15000

20000

25000

Samples

Ne

t S

ign

al In

ten

sit

y (

MF

I)

miR-9

miR-122

miR-1






289704

12431797

4050

7044

12899

18030

24973

23884

21852

19249

11326

6573

3852

2471

0

5000

10000

15000

20000

25000

30000


Input Amount

Ne

t S

ign

al In

ten

sit

y (

MF

I)

200 amol


81


0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000



Ne

t S

ign

al in

ten

sit

y (

MF

I)

82

miR-133a

000

020

040

060

080

100

120


Total RNA Samples

Rela

tive C

han

ge t

o H

eart

FlexmiR v2

qRT-PCR

Tissue

Re

lati

ve

Ch

an

ge


LNP-miRNA

qRT-PCR

83

miRNA

Re

lati

ve

Ch

an

ge



0

5

10

15

20

25


miRNA

Rela

tive C

han

ge

FlexmiR v2

qRT-PCR

LNP-miRNA

qPCR

84



0

5000

10000

15000

20000

25000

30000

hsa-m

iR-1

07

hsa-m

iR-1

47

hsa-m

iR-1

81a

hsa-m

iR-1

92

hsa-m

iR-1

9b

hsa-m

iR-2

20a

hsa-m

iR-2

1hsa-m

iR-2

18

hsa-le

t-7c

hsa-m

iR-1

00

hsa-m

iR-1

25b

hsa-m

iR-

hsa-m

iR-2

05

hsa-m

iR-2

21

hsa-m

iR-2

00a

hsa-m

iR-2

00c

hsa-m

iR-2

06

hsa-m

iR-1

0a

hsa-m

iR-1

6hsa-m

iR-9

hsa-m

iR-4

24

hsa-m

iR-2

0a

hsa-m

iR-2

0b

hsa-le

t-7a

hsa-m

iR-9

5hsa-m

iR-1

36

hsa-m

iR-1

95

hsa-m

iR-2

10

hsa-m

iR-2

12

hsa-m

iR-

hsa-m

iR-1

7hsa-m

iR-1

22

hsa-m

iR-1

43

hsa-m

iR-1

45

hsa-m

ir-1

06a

hsa-m

iR-

hsa-m

iR-1

0b

hsa-m

iR-1

hsa-m

iR-2

23

hsa-m

iR-1

81b

hsa-le

t-155

hsa-le

t-7g

hsa-m

iR-7

ihsa-m

iR-9

6hsa-m

iR-1

37

hsa-m

iR-1

85

hsa-m

iR-1

38

hsa-m

iR-1

33a

hsa-m

iR-1

33b

hsa-m

iR-1

5a

hsa-m

iR-3

4a

hsa-m

iR-2

9a

hsa-m

iR-2

9b

hsa-m

iR-1

03

hsa-m

iR-2

22

hsa-m

iR-1

46a

hsa-m

iR-3

9cel-m

iR-3

9cel-m

iR-2

38

Regions

Ne

t S

ign

al in

ten

sit

y (

MF

I)

50ng

100ng

250ng

500ng








bull Flexible





Proteomic Assays



Nucleic Acids








or IgM





Multiplex Detection

Detection


IgG

1 2 3 4





















Y Y

Y

IgG1 IgG2

IgG3

IgG4 IgA



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Detection



Antigen

1 2 3








Y Y Y

Y

Y

Y Y

Y Y

Y Y

Y



Y


Detection

Multiplex Detection


















compounds





compounds



SAPE SAPE














SAPE SAPE


Detection

Multiplex Detection












543-550






GTPases 827 271-282














Single Plex Assays

To Luminex

Multiplex

Assays










Detection

Sequence Detection

TAG


















Workflow

75



2 hours



30 minutes



30 minutes





30 minutes

76






0

500

1000

1500

2000

2500

3000

3500

4000


Sample

Sig

nal In

ten

sit

y (

MF

I)

let-7c

Let-7a

77


miR-107 vs miR-103

0

1000

2000

3000

4000

5000

6000


Sample

Sig

na

l In

ten

sit

y (

MF

I)

miR-103

miR-107









0

1000

2000

3000

4000

5000

6000

7000

8000

9000

10000

hsa-m

iR-1

07

hsa-m

iR-1

47

hsa-m

iR-1

81a

hsa-m

iR-1

92

hsa-m

iR-1

9b

hsa-m

iR-2

20a

hsa-m

iR-2

1hsa-m

iR-2

18

hsa-le

t-7c

hsa-m

iR-1

00

hsa-m

iR-1

25b

hsa-m

iR-

hsa-m

iR-2

05

hsa-m

iR-2

21

hsa-m

iR-2

00a

hsa-m

iR-2

00c

hsa-m

iR-2

06

hsa-m

iR-1

0a

hsa-m

iR-1

6

hsa-m

iR-9

hsa-m

iR-4

24

hsa-m

iR-2

0a

hsa-m

iR-2

0b

hsa-le

t-7a

hsa-m

iR-9

5hsa-m

iR-1

36

hsa-m

iR-1

95

hsa-m

iR-2

10

hsa-m

iR-2

12

hsa-m

iR-

hsa-m

iR-1

7

hsa-m

iR-1

22

hsa-m

iR-1

43

hsa-m

iR-1

45

hsa-m

iR-1

06a

hsa-m

iR-

hsa-m

iR-1

0b

hsa-m

iR-1

hsa-m

iR-2

23

hsa-m

iR-1

81b

hsa-m

iR-1

55

hsa-le

t-7g

hsa-le

t-7i

hsa-m

iR-9

6

hsa-m

iR-1

37

hsa-m

iR-1

85

hsa-m

iR-1

38

hsa-m

iR-1

98

hsa-m

iR-1

33a

hsa-m

iR-1

33b

hsa-m

iR-1

5a

hsa-m

iR-3

4a

hsa-m

iR-2

9a

hsa-m

iR-2

9b

hsa-m

iR-1

03

hsa-m

iR-2

22

hsa-m

iR-1

46a

cel-m

iR-3

9cel-m

iR-5

4

cel-m

iR-2

38

Regions

Ne

t S

ign

al In

ten

sit

y (

MF

I)

106a

17


Total RNA Samples

53-Plex Assay

miR-9 miR-122 miR-1

0

5000

10000

15000

20000

25000

Samples

Ne

t S

ign

al In

ten

sit

y (

MF

I)

miR-9

miR-122

miR-1






289704

12431797

4050

7044

12899

18030

24973

23884

21852

19249

11326

6573

3852

2471

0

5000

10000

15000

20000

25000

30000


Input Amount

Ne

t S

ign

al In

ten

sit

y (

MF

I)

200 amol


81


0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000



Ne

t S

ign

al in

ten

sit

y (

MF

I)

82

miR-133a

000

020

040

060

080

100

120


Total RNA Samples

Rela

tive C

han

ge t

o H

eart

FlexmiR v2

qRT-PCR

Tissue

Re

lati

ve

Ch

an

ge


LNP-miRNA

qRT-PCR

83

miRNA

Re

lati

ve

Ch

an

ge



0

5

10

15

20

25


miRNA

Rela

tive C

han

ge

FlexmiR v2

qRT-PCR

LNP-miRNA

qPCR

84



0

5000

10000

15000

20000

25000

30000

hsa-m

iR-1

07

hsa-m

iR-1

47

hsa-m

iR-1

81a

hsa-m

iR-1

92

hsa-m

iR-1

9b

hsa-m

iR-2

20a

hsa-m

iR-2

1hsa-m

iR-2

18

hsa-le

t-7c

hsa-m

iR-1

00

hsa-m

iR-1

25b

hsa-m

iR-

hsa-m

iR-2

05

hsa-m

iR-2

21

hsa-m

iR-2

00a

hsa-m

iR-2

00c

hsa-m

iR-2

06

hsa-m

iR-1

0a

hsa-m

iR-1

6hsa-m

iR-9

hsa-m

iR-4

24

hsa-m

iR-2

0a

hsa-m

iR-2

0b

hsa-le

t-7a

hsa-m

iR-9

5hsa-m

iR-1

36

hsa-m

iR-1

95

hsa-m

iR-2

10

hsa-m

iR-2

12

hsa-m

iR-

hsa-m

iR-1

7hsa-m

iR-1

22

hsa-m

iR-1

43

hsa-m

iR-1

45

hsa-m

ir-1

06a

hsa-m

iR-

hsa-m

iR-1

0b

hsa-m

iR-1

hsa-m

iR-2

23

hsa-m

iR-1

81b

hsa-le

t-155

hsa-le

t-7g

hsa-m

iR-7

ihsa-m

iR-9

6hsa-m

iR-1

37

hsa-m

iR-1

85

hsa-m

iR-1

38

hsa-m

iR-1

33a

hsa-m

iR-1

33b

hsa-m

iR-1

5a

hsa-m

iR-3

4a

hsa-m

iR-2

9a

hsa-m

iR-2

9b

hsa-m

iR-1

03

hsa-m

iR-2

22

hsa-m

iR-1

46a

hsa-m

iR-3

9cel-m

iR-3

9cel-m

iR-2

38

Regions

Ne

t S

ign

al in

ten

sit

y (

MF

I)

50ng

100ng

250ng

500ng








bull Flexible





Proteomic Assays



Nucleic Acids








or IgM





Multiplex Detection

Detection


IgG

1 2 3 4





















Y Y

Y

IgG1 IgG2

IgG3

IgG4 IgA



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Detection



Antigen

1 2 3








Y Y Y

Y

Y

Y Y

Y Y

Y Y

Y



Y


Detection

Multiplex Detection


















compounds





compounds



SAPE SAPE














SAPE SAPE


Detection

Multiplex Detection












543-550






GTPases 827 271-282














Single Plex Assays

To Luminex

Multiplex

Assays








Detection

Sequence Detection

TAG


















Workflow

75



2 hours



30 minutes



30 minutes





30 minutes

76






0

500

1000

1500

2000

2500

3000

3500

4000


Sample

Sig

nal In

ten

sit

y (

MF

I)

let-7c

Let-7a

77


miR-107 vs miR-103

0

1000

2000

3000

4000

5000

6000


Sample

Sig

na

l In

ten

sit

y (

MF

I)

miR-103

miR-107









0

1000

2000

3000

4000

5000

6000

7000

8000

9000

10000

hsa-m

iR-1

07

hsa-m

iR-1

47

hsa-m

iR-1

81a

hsa-m

iR-1

92

hsa-m

iR-1

9b

hsa-m

iR-2

20a

hsa-m

iR-2

1hsa-m

iR-2

18

hsa-le

t-7c

hsa-m

iR-1

00

hsa-m

iR-1

25b

hsa-m

iR-

hsa-m

iR-2

05

hsa-m

iR-2

21

hsa-m

iR-2

00a

hsa-m

iR-2

00c

hsa-m

iR-2

06

hsa-m

iR-1

0a

hsa-m

iR-1

6

hsa-m

iR-9

hsa-m

iR-4

24

hsa-m

iR-2

0a

hsa-m

iR-2

0b

hsa-le

t-7a

hsa-m

iR-9

5hsa-m

iR-1

36

hsa-m

iR-1

95

hsa-m

iR-2

10

hsa-m

iR-2

12

hsa-m

iR-

hsa-m

iR-1

7

hsa-m

iR-1

22

hsa-m

iR-1

43

hsa-m

iR-1

45

hsa-m

iR-1

06a

hsa-m

iR-

hsa-m

iR-1

0b

hsa-m

iR-1

hsa-m

iR-2

23

hsa-m

iR-1

81b

hsa-m

iR-1

55

hsa-le

t-7g

hsa-le

t-7i

hsa-m

iR-9

6

hsa-m

iR-1

37

hsa-m

iR-1

85

hsa-m

iR-1

38

hsa-m

iR-1

98

hsa-m

iR-1

33a

hsa-m

iR-1

33b

hsa-m

iR-1

5a

hsa-m

iR-3

4a

hsa-m

iR-2

9a

hsa-m

iR-2

9b

hsa-m

iR-1

03

hsa-m

iR-2

22

hsa-m

iR-1

46a

cel-m

iR-3

9cel-m

iR-5

4

cel-m

iR-2

38

Regions

Ne

t S

ign

al In

ten

sit

y (

MF

I)

106a

17


Total RNA Samples

53-Plex Assay

miR-9 miR-122 miR-1

0

5000

10000

15000

20000

25000

Samples

Ne

t S

ign

al In

ten

sit

y (

MF

I)

miR-9

miR-122

miR-1






289704

12431797

4050

7044

12899

18030

24973

23884

21852

19249

11326

6573

3852

2471

0

5000

10000

15000

20000

25000

30000


Input Amount

Ne

t S

ign

al In

ten

sit

y (

MF

I)

200 amol


81


0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000



Ne

t S

ign

al in

ten

sit

y (

MF

I)

82

miR-133a

000

020

040

060

080

100

120


Total RNA Samples

Rela

tive C

han

ge t

o H

eart

FlexmiR v2

qRT-PCR

Tissue

Re

lati

ve

Ch

an

ge


LNP-miRNA

qRT-PCR

83

miRNA

Re

lati

ve

Ch

an

ge



0

5

10

15

20

25


miRNA

Rela

tive C

han

ge

FlexmiR v2

qRT-PCR

LNP-miRNA

qPCR

84



0

5000

10000

15000

20000

25000

30000

hsa-m

iR-1

07

hsa-m

iR-1

47

hsa-m

iR-1

81a

hsa-m

iR-1

92

hsa-m

iR-1

9b

hsa-m

iR-2

20a

hsa-m

iR-2

1hsa-m

iR-2

18

hsa-le

t-7c

hsa-m

iR-1

00

hsa-m

iR-1

25b

hsa-m

iR-

hsa-m

iR-2

05

hsa-m

iR-2

21

hsa-m

iR-2

00a

hsa-m

iR-2

00c

hsa-m

iR-2

06

hsa-m

iR-1

0a

hsa-m

iR-1

6hsa-m

iR-9

hsa-m

iR-4

24

hsa-m

iR-2

0a

hsa-m

iR-2

0b

hsa-le

t-7a

hsa-m

iR-9

5hsa-m

iR-1

36

hsa-m

iR-1

95

hsa-m

iR-2

10

hsa-m

iR-2

12

hsa-m

iR-

hsa-m

iR-1

7hsa-m

iR-1

22

hsa-m

iR-1

43

hsa-m

iR-1

45

hsa-m

ir-1

06a

hsa-m

iR-

hsa-m

iR-1

0b

hsa-m

iR-1

hsa-m

iR-2

23

hsa-m

iR-1

81b

hsa-le

t-155

hsa-le

t-7g

hsa-m

iR-7

ihsa-m

iR-9

6hsa-m

iR-1

37

hsa-m

iR-1

85

hsa-m

iR-1

38

hsa-m

iR-1

33a

hsa-m

iR-1

33b

hsa-m

iR-1

5a

hsa-m

iR-3

4a

hsa-m

iR-2

9a

hsa-m

iR-2

9b

hsa-m

iR-1

03

hsa-m

iR-2

22

hsa-m

iR-1

46a

hsa-m

iR-3

9cel-m

iR-3

9cel-m

iR-2

38

Regions

Ne

t S

ign

al in

ten

sit

y (

MF

I)

50ng

100ng

250ng

500ng








bull Flexible





Proteomic Assays



Nucleic Acids








or IgM





Multiplex Detection

Detection


IgG

1 2 3 4





















Y Y

Y

IgG1 IgG2

IgG3

IgG4 IgA



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Detection



Antigen

1 2 3








Y Y Y

Y

Y

Y Y

Y Y

Y Y

Y



Y


Detection

Multiplex Detection


















compounds





compounds



SAPE SAPE














SAPE SAPE


Detection

Multiplex Detection












543-550






GTPases 827 271-282














Single Plex Assays

To Luminex

Multiplex

Assays




















Workflow

75



2 hours



30 minutes



30 minutes





30 minutes

76






0

500

1000

1500

2000

2500

3000

3500

4000


Sample

Sig

nal In

ten

sit

y (

MF

I)

let-7c

Let-7a

77


miR-107 vs miR-103

0

1000

2000

3000

4000

5000

6000


Sample

Sig

na

l In

ten

sit

y (

MF

I)

miR-103

miR-107









0

1000

2000

3000

4000

5000

6000

7000

8000

9000

10000

hsa-m

iR-1

07

hsa-m

iR-1

47

hsa-m

iR-1

81a

hsa-m

iR-1

92

hsa-m

iR-1

9b

hsa-m

iR-2

20a

hsa-m

iR-2

1hsa-m

iR-2

18

hsa-le

t-7c

hsa-m

iR-1

00

hsa-m

iR-1

25b

hsa-m

iR-

hsa-m

iR-2

05

hsa-m

iR-2

21

hsa-m

iR-2

00a

hsa-m

iR-2

00c

hsa-m

iR-2

06

hsa-m

iR-1

0a

hsa-m

iR-1

6

hsa-m

iR-9

hsa-m

iR-4

24

hsa-m

iR-2

0a

hsa-m

iR-2

0b

hsa-le

t-7a

hsa-m

iR-9

5hsa-m

iR-1

36

hsa-m

iR-1

95

hsa-m

iR-2

10

hsa-m

iR-2

12

hsa-m

iR-

hsa-m

iR-1

7

hsa-m

iR-1

22

hsa-m

iR-1

43

hsa-m

iR-1

45

hsa-m

iR-1

06a

hsa-m

iR-

hsa-m

iR-1

0b

hsa-m

iR-1

hsa-m

iR-2

23

hsa-m

iR-1

81b

hsa-m

iR-1

55

hsa-le

t-7g

hsa-le

t-7i

hsa-m

iR-9

6

hsa-m

iR-1

37

hsa-m

iR-1

85

hsa-m

iR-1

38

hsa-m

iR-1

98

hsa-m

iR-1

33a

hsa-m

iR-1

33b

hsa-m

iR-1

5a

hsa-m

iR-3

4a

hsa-m

iR-2

9a

hsa-m

iR-2

9b

hsa-m

iR-1

03

hsa-m

iR-2

22

hsa-m

iR-1

46a

cel-m

iR-3

9cel-m

iR-5

4

cel-m

iR-2

38

Regions

Ne

t S

ign

al In

ten

sit

y (

MF

I)

106a

17


Total RNA Samples

53-Plex Assay

miR-9 miR-122 miR-1

0

5000

10000

15000

20000

25000

Samples

Ne

t S

ign

al In

ten

sit

y (

MF

I)

miR-9

miR-122

miR-1






289704

12431797

4050

7044

12899

18030

24973

23884

21852

19249

11326

6573

3852

2471

0

5000

10000

15000

20000

25000

30000


Input Amount

Ne

t S

ign

al In

ten

sit

y (

MF

I)

200 amol


81


0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000



Ne

t S

ign

al in

ten

sit

y (

MF

I)

82

miR-133a

000

020

040

060

080

100

120


Total RNA Samples

Rela

tive C

han

ge t

o H

eart

FlexmiR v2

qRT-PCR

Tissue

Re

lati

ve

Ch

an

ge


LNP-miRNA

qRT-PCR

83

miRNA

Re

lati

ve

Ch

an

ge



0

5

10

15

20

25


miRNA

Rela

tive C

han

ge

FlexmiR v2

qRT-PCR

LNP-miRNA

qPCR

84



0

5000

10000

15000

20000

25000

30000

hsa-m

iR-1

07

hsa-m

iR-1

47

hsa-m

iR-1

81a

hsa-m

iR-1

92

hsa-m

iR-1

9b

hsa-m

iR-2

20a

hsa-m

iR-2

1hsa-m

iR-2

18

hsa-le

t-7c

hsa-m

iR-1

00

hsa-m

iR-1

25b

hsa-m

iR-

hsa-m

iR-2

05

hsa-m

iR-2

21

hsa-m

iR-2

00a

hsa-m

iR-2

00c

hsa-m

iR-2

06

hsa-m

iR-1

0a

hsa-m

iR-1

6hsa-m

iR-9

hsa-m

iR-4

24

hsa-m

iR-2

0a

hsa-m

iR-2

0b

hsa-le

t-7a

hsa-m

iR-9

5hsa-m

iR-1

36

hsa-m

iR-1

95

hsa-m

iR-2

10

hsa-m

iR-2

12

hsa-m

iR-

hsa-m

iR-1

7hsa-m

iR-1

22

hsa-m

iR-1

43

hsa-m

iR-1

45

hsa-m

ir-1

06a

hsa-m

iR-

hsa-m

iR-1

0b

hsa-m

iR-1

hsa-m

iR-2

23

hsa-m

iR-1

81b

hsa-le

t-155

hsa-le

t-7g

hsa-m

iR-7

ihsa-m

iR-9

6hsa-m

iR-1

37

hsa-m

iR-1

85

hsa-m

iR-1

38

hsa-m

iR-1

33a

hsa-m

iR-1

33b

hsa-m

iR-1

5a

hsa-m

iR-3

4a

hsa-m

iR-2

9a

hsa-m

iR-2

9b

hsa-m

iR-1

03

hsa-m

iR-2

22

hsa-m

iR-1

46a

hsa-m

iR-3

9cel-m

iR-3

9cel-m

iR-2

38

Regions

Ne

t S

ign

al in

ten

sit

y (

MF

I)

50ng

100ng

250ng

500ng








bull Flexible





Proteomic Assays



Nucleic Acids








or IgM





Multiplex Detection

Detection


IgG

1 2 3 4





















Y Y

Y

IgG1 IgG2

IgG3

IgG4 IgA



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Detection



Antigen

1 2 3








Y Y Y

Y

Y

Y Y

Y Y

Y Y

Y



Y


Detection

Multiplex Detection


















compounds





compounds



SAPE SAPE














SAPE SAPE


Detection

Multiplex Detection












543-550






GTPases 827 271-282














Single Plex Assays

To Luminex

Multiplex

Assays












Workflow

75



2 hours



30 minutes



30 minutes





30 minutes

76






0

500

1000

1500

2000

2500

3000

3500

4000


Sample

Sig

nal In

ten

sit

y (

MF

I)

let-7c

Let-7a

77


miR-107 vs miR-103

0

1000

2000

3000

4000

5000

6000


Sample

Sig

na

l In

ten

sit

y (

MF

I)

miR-103

miR-107









0

1000

2000

3000

4000

5000

6000

7000

8000

9000

10000

hsa-m

iR-1

07

hsa-m

iR-1

47

hsa-m

iR-1

81a

hsa-m

iR-1

92

hsa-m

iR-1

9b

hsa-m

iR-2

20a

hsa-m

iR-2

1hsa-m

iR-2

18

hsa-le

t-7c

hsa-m

iR-1

00

hsa-m

iR-1

25b

hsa-m

iR-

hsa-m

iR-2

05

hsa-m

iR-2

21

hsa-m

iR-2

00a

hsa-m

iR-2

00c

hsa-m

iR-2

06

hsa-m

iR-1

0a

hsa-m

iR-1

6

hsa-m

iR-9

hsa-m

iR-4

24

hsa-m

iR-2

0a

hsa-m

iR-2

0b

hsa-le

t-7a

hsa-m

iR-9

5hsa-m

iR-1

36

hsa-m

iR-1

95

hsa-m

iR-2

10

hsa-m

iR-2

12

hsa-m

iR-

hsa-m

iR-1

7

hsa-m

iR-1

22

hsa-m

iR-1

43

hsa-m

iR-1

45

hsa-m

iR-1

06a

hsa-m

iR-

hsa-m

iR-1

0b

hsa-m

iR-1

hsa-m

iR-2

23

hsa-m

iR-1

81b

hsa-m

iR-1

55

hsa-le

t-7g

hsa-le

t-7i

hsa-m

iR-9

6

hsa-m

iR-1

37

hsa-m

iR-1

85

hsa-m

iR-1

38

hsa-m

iR-1

98

hsa-m

iR-1

33a

hsa-m

iR-1

33b

hsa-m

iR-1

5a

hsa-m

iR-3

4a

hsa-m

iR-2

9a

hsa-m

iR-2

9b

hsa-m

iR-1

03

hsa-m

iR-2

22

hsa-m

iR-1

46a

cel-m

iR-3

9cel-m

iR-5

4

cel-m

iR-2

38

Regions

Ne

t S

ign

al In

ten

sit

y (

MF

I)

106a

17


Total RNA Samples

53-Plex Assay

miR-9 miR-122 miR-1

0

5000

10000

15000

20000

25000

Samples

Ne

t S

ign

al In

ten

sit

y (

MF

I)

miR-9

miR-122

miR-1






289704

12431797

4050

7044

12899

18030

24973

23884

21852

19249

11326

6573

3852

2471

0

5000

10000

15000

20000

25000

30000


Input Amount

Ne

t S

ign

al In

ten

sit

y (

MF

I)

200 amol


81


0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000



Ne

t S

ign

al in

ten

sit

y (

MF

I)

82

miR-133a

000

020

040

060

080

100

120


Total RNA Samples

Rela

tive C

han

ge t

o H

eart

FlexmiR v2

qRT-PCR

Tissue

Re

lati

ve

Ch

an

ge


LNP-miRNA

qRT-PCR

83

miRNA

Re

lati

ve

Ch

an

ge



0

5

10

15

20

25


miRNA

Rela

tive C

han

ge

FlexmiR v2

qRT-PCR

LNP-miRNA

qPCR

84



0

5000

10000

15000

20000

25000

30000

hsa-m

iR-1

07

hsa-m

iR-1

47

hsa-m

iR-1

81a

hsa-m

iR-1

92

hsa-m

iR-1

9b

hsa-m

iR-2

20a

hsa-m

iR-2

1hsa-m

iR-2

18

hsa-le

t-7c

hsa-m

iR-1

00

hsa-m

iR-1

25b

hsa-m

iR-

hsa-m

iR-2

05

hsa-m

iR-2

21

hsa-m

iR-2

00a

hsa-m

iR-2

00c

hsa-m

iR-2

06

hsa-m

iR-1

0a

hsa-m

iR-1

6hsa-m

iR-9

hsa-m

iR-4

24

hsa-m

iR-2

0a

hsa-m

iR-2

0b

hsa-le

t-7a

hsa-m

iR-9

5hsa-m

iR-1

36

hsa-m

iR-1

95

hsa-m

iR-2

10

hsa-m

iR-2

12

hsa-m

iR-

hsa-m

iR-1

7hsa-m

iR-1

22

hsa-m

iR-1

43

hsa-m

iR-1

45

hsa-m

ir-1

06a

hsa-m

iR-

hsa-m

iR-1

0b

hsa-m

iR-1

hsa-m

iR-2

23

hsa-m

iR-1

81b

hsa-le

t-155

hsa-le

t-7g

hsa-m

iR-7

ihsa-m

iR-9

6hsa-m

iR-1

37

hsa-m

iR-1

85

hsa-m

iR-1

38

hsa-m

iR-1

33a

hsa-m

iR-1

33b

hsa-m

iR-1

5a

hsa-m

iR-3

4a

hsa-m

iR-2

9a

hsa-m

iR-2

9b

hsa-m

iR-1

03

hsa-m

iR-2

22

hsa-m

iR-1

46a

hsa-m

iR-3

9cel-m

iR-3

9cel-m

iR-2

38

Regions

Ne

t S

ign

al in

ten

sit

y (

MF

I)

50ng

100ng

250ng

500ng








bull Flexible





Proteomic Assays



Nucleic Acids








or IgM





Multiplex Detection

Detection


IgG

1 2 3 4





















Y Y

Y

IgG1 IgG2

IgG3

IgG4 IgA



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Detection



Antigen

1 2 3








Y Y Y

Y

Y

Y Y

Y Y

Y Y

Y



Y


Detection

Multiplex Detection


















compounds





compounds



SAPE SAPE














SAPE SAPE


Detection

Multiplex Detection












543-550






GTPases 827 271-282














Single Plex Assays

To Luminex

Multiplex

Assays











Workflow

75



2 hours



30 minutes



30 minutes





30 minutes

76






0

500

1000

1500

2000

2500

3000

3500

4000


Sample

Sig

nal In

ten

sit

y (

MF

I)

let-7c

Let-7a

77


miR-107 vs miR-103

0

1000

2000

3000

4000

5000

6000


Sample

Sig

na

l In

ten

sit

y (

MF

I)

miR-103

miR-107









0

1000

2000

3000

4000

5000

6000

7000

8000

9000

10000

hsa-m

iR-1

07

hsa-m

iR-1

47

hsa-m

iR-1

81a

hsa-m

iR-1

92

hsa-m

iR-1

9b

hsa-m

iR-2

20a

hsa-m

iR-2

1hsa-m

iR-2

18

hsa-le

t-7c

hsa-m

iR-1

00

hsa-m

iR-1

25b

hsa-m

iR-

hsa-m

iR-2

05

hsa-m

iR-2

21

hsa-m

iR-2

00a

hsa-m

iR-2

00c

hsa-m

iR-2

06

hsa-m

iR-1

0a

hsa-m

iR-1

6

hsa-m

iR-9

hsa-m

iR-4

24

hsa-m

iR-2

0a

hsa-m

iR-2

0b

hsa-le

t-7a

hsa-m

iR-9

5hsa-m

iR-1

36

hsa-m

iR-1

95

hsa-m

iR-2

10

hsa-m

iR-2

12

hsa-m

iR-

hsa-m

iR-1

7

hsa-m

iR-1

22

hsa-m

iR-1

43

hsa-m

iR-1

45

hsa-m

iR-1

06a

hsa-m

iR-

hsa-m

iR-1

0b

hsa-m

iR-1

hsa-m

iR-2

23

hsa-m

iR-1

81b

hsa-m

iR-1

55

hsa-le

t-7g

hsa-le

t-7i

hsa-m

iR-9

6

hsa-m

iR-1

37

hsa-m

iR-1

85

hsa-m

iR-1

38

hsa-m

iR-1

98

hsa-m

iR-1

33a

hsa-m

iR-1

33b

hsa-m

iR-1

5a

hsa-m

iR-3

4a

hsa-m

iR-2

9a

hsa-m

iR-2

9b

hsa-m

iR-1

03

hsa-m

iR-2

22

hsa-m

iR-1

46a

cel-m

iR-3

9cel-m

iR-5

4

cel-m

iR-2

38

Regions

Ne

t S

ign

al In

ten

sit

y (

MF

I)

106a

17


Total RNA Samples

53-Plex Assay

miR-9 miR-122 miR-1

0

5000

10000

15000

20000

25000

Samples

Ne

t S

ign

al In

ten

sit

y (

MF

I)

miR-9

miR-122

miR-1






289704

12431797

4050

7044

12899

18030

24973

23884

21852

19249

11326

6573

3852

2471

0

5000

10000

15000

20000

25000

30000


Input Amount

Ne

t S

ign

al In

ten

sit

y (

MF

I)

200 amol


81


0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000



Ne

t S

ign

al in

ten

sit

y (

MF

I)

82

miR-133a

000

020

040

060

080

100

120


Total RNA Samples

Rela

tive C

han

ge t

o H

eart

FlexmiR v2

qRT-PCR

Tissue

Re

lati

ve

Ch

an

ge


LNP-miRNA

qRT-PCR

83

miRNA

Re

lati

ve

Ch

an

ge



0

5

10

15

20

25


miRNA

Rela

tive C

han

ge

FlexmiR v2

qRT-PCR

LNP-miRNA

qPCR

84



0

5000

10000

15000

20000

25000

30000

hsa-m

iR-1

07

hsa-m

iR-1

47

hsa-m

iR-1

81a

hsa-m

iR-1

92

hsa-m

iR-1

9b

hsa-m

iR-2

20a

hsa-m

iR-2

1hsa-m

iR-2

18

hsa-le

t-7c

hsa-m

iR-1

00

hsa-m

iR-1

25b

hsa-m

iR-

hsa-m

iR-2

05

hsa-m

iR-2

21

hsa-m

iR-2

00a

hsa-m

iR-2

00c

hsa-m

iR-2

06

hsa-m

iR-1

0a

hsa-m

iR-1

6hsa-m

iR-9

hsa-m

iR-4

24

hsa-m

iR-2

0a

hsa-m

iR-2

0b

hsa-le

t-7a

hsa-m

iR-9

5hsa-m

iR-1

36

hsa-m

iR-1

95

hsa-m

iR-2

10

hsa-m

iR-2

12

hsa-m

iR-

hsa-m

iR-1

7hsa-m

iR-1

22

hsa-m

iR-1

43

hsa-m

iR-1

45

hsa-m

ir-1

06a

hsa-m

iR-

hsa-m

iR-1

0b

hsa-m

iR-1

hsa-m

iR-2

23

hsa-m

iR-1

81b

hsa-le

t-155

hsa-le

t-7g

hsa-m

iR-7

ihsa-m

iR-9

6hsa-m

iR-1

37

hsa-m

iR-1

85

hsa-m

iR-1

38

hsa-m

iR-1

33a

hsa-m

iR-1

33b

hsa-m

iR-1

5a

hsa-m

iR-3

4a

hsa-m

iR-2

9a

hsa-m

iR-2

9b

hsa-m

iR-1

03

hsa-m

iR-2

22

hsa-m

iR-1

46a

hsa-m

iR-3

9cel-m

iR-3

9cel-m

iR-2

38

Regions

Ne

t S

ign

al in

ten

sit

y (

MF

I)

50ng

100ng

250ng

500ng








bull Flexible





Proteomic Assays



Nucleic Acids








or IgM





Multiplex Detection

Detection


IgG

1 2 3 4





















Y Y

Y

IgG1 IgG2

IgG3

IgG4 IgA



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Detection



Antigen

1 2 3








Y Y Y

Y

Y

Y Y

Y Y

Y Y

Y



Y


Detection

Multiplex Detection


















compounds





compounds



SAPE SAPE














SAPE SAPE


Detection

Multiplex Detection












543-550






GTPases 827 271-282














Single Plex Assays

To Luminex

Multiplex

Assays



Workflow

75



2 hours



30 minutes



30 minutes





30 minutes

76






0

500

1000

1500

2000

2500

3000

3500

4000


Sample

Sig

nal In

ten

sit

y (

MF

I)

let-7c

Let-7a

77


miR-107 vs miR-103

0

1000

2000

3000

4000

5000

6000


Sample

Sig

na

l In

ten

sit

y (

MF

I)

miR-103

miR-107









0

1000

2000

3000

4000

5000

6000

7000

8000

9000

10000

hsa-m

iR-1

07

hsa-m

iR-1

47

hsa-m

iR-1

81a

hsa-m

iR-1

92

hsa-m

iR-1

9b

hsa-m

iR-2

20a

hsa-m

iR-2

1hsa-m

iR-2

18

hsa-le

t-7c

hsa-m

iR-1

00

hsa-m

iR-1

25b

hsa-m

iR-

hsa-m

iR-2

05

hsa-m

iR-2

21

hsa-m

iR-2

00a

hsa-m

iR-2

00c

hsa-m

iR-2

06

hsa-m

iR-1

0a

hsa-m

iR-1

6

hsa-m

iR-9

hsa-m

iR-4

24

hsa-m

iR-2

0a

hsa-m

iR-2

0b

hsa-le

t-7a

hsa-m

iR-9

5hsa-m

iR-1

36

hsa-m

iR-1

95

hsa-m

iR-2

10

hsa-m

iR-2

12

hsa-m

iR-

hsa-m

iR-1

7

hsa-m

iR-1

22

hsa-m

iR-1

43

hsa-m

iR-1

45

hsa-m

iR-1

06a

hsa-m

iR-

hsa-m

iR-1

0b

hsa-m

iR-1

hsa-m

iR-2

23

hsa-m

iR-1

81b

hsa-m

iR-1

55

hsa-le

t-7g

hsa-le

t-7i

hsa-m

iR-9

6

hsa-m

iR-1

37

hsa-m

iR-1

85

hsa-m

iR-1

38

hsa-m

iR-1

98

hsa-m

iR-1

33a

hsa-m

iR-1

33b

hsa-m

iR-1

5a

hsa-m

iR-3

4a

hsa-m

iR-2

9a

hsa-m

iR-2

9b

hsa-m

iR-1

03

hsa-m

iR-2

22

hsa-m

iR-1

46a

cel-m

iR-3

9cel-m

iR-5

4

cel-m

iR-2

38

Regions

Ne

t S

ign

al In

ten

sit

y (

MF

I)

106a

17


Total RNA Samples

53-Plex Assay

miR-9 miR-122 miR-1

0

5000

10000

15000

20000

25000

Samples

Ne

t S

ign

al In

ten

sit

y (

MF

I)

miR-9

miR-122

miR-1






289704

12431797

4050

7044

12899

18030

24973

23884

21852

19249

11326

6573

3852

2471

0

5000

10000

15000

20000

25000

30000


Input Amount

Ne

t S

ign

al In

ten

sit

y (

MF

I)

200 amol


81


0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000



Ne

t S

ign

al in

ten

sit

y (

MF

I)

82

miR-133a

000

020

040

060

080

100

120


Total RNA Samples

Rela

tive C

han

ge t

o H

eart

FlexmiR v2

qRT-PCR

Tissue

Re

lati

ve

Ch

an

ge


LNP-miRNA

qRT-PCR

83

miRNA

Re

lati

ve

Ch

an

ge



0

5

10

15

20

25


miRNA

Rela

tive C

han

ge

FlexmiR v2

qRT-PCR

LNP-miRNA

qPCR

84



0

5000

10000

15000

20000

25000

30000

hsa-m

iR-1

07

hsa-m

iR-1

47

hsa-m

iR-1

81a

hsa-m

iR-1

92

hsa-m

iR-1

9b

hsa-m

iR-2

20a

hsa-m

iR-2

1hsa-m

iR-2

18

hsa-le

t-7c

hsa-m

iR-1

00

hsa-m

iR-1

25b

hsa-m

iR-

hsa-m

iR-2

05

hsa-m

iR-2

21

hsa-m

iR-2

00a

hsa-m

iR-2

00c

hsa-m

iR-2

06

hsa-m

iR-1

0a

hsa-m

iR-1

6hsa-m

iR-9

hsa-m

iR-4

24

hsa-m

iR-2

0a

hsa-m

iR-2

0b

hsa-le

t-7a

hsa-m

iR-9

5hsa-m

iR-1

36

hsa-m

iR-1

95

hsa-m

iR-2

10

hsa-m

iR-2

12

hsa-m

iR-

hsa-m

iR-1

7hsa-m

iR-1

22

hsa-m

iR-1

43

hsa-m

iR-1

45

hsa-m

ir-1

06a

hsa-m

iR-

hsa-m

iR-1

0b

hsa-m

iR-1

hsa-m

iR-2

23

hsa-m

iR-1

81b

hsa-le

t-155

hsa-le

t-7g

hsa-m

iR-7

ihsa-m

iR-9

6hsa-m

iR-1

37

hsa-m

iR-1

85

hsa-m

iR-1

38

hsa-m

iR-1

33a

hsa-m

iR-1

33b

hsa-m

iR-1

5a

hsa-m

iR-3

4a

hsa-m

iR-2

9a

hsa-m

iR-2

9b

hsa-m

iR-1

03

hsa-m

iR-2

22

hsa-m

iR-1

46a

hsa-m

iR-3

9cel-m

iR-3

9cel-m

iR-2

38

Regions

Ne

t S

ign

al in

ten

sit

y (

MF

I)

50ng

100ng

250ng

500ng








bull Flexible





Proteomic Assays



Nucleic Acids








or IgM





Multiplex Detection

Detection


IgG

1 2 3 4





















Y Y

Y

IgG1 IgG2

IgG3

IgG4 IgA



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Detection



Antigen

1 2 3








Y Y Y

Y

Y

Y Y

Y Y

Y Y

Y



Y


Detection

Multiplex Detection


















compounds





compounds



SAPE SAPE














SAPE SAPE


Detection

Multiplex Detection












543-550






GTPases 827 271-282














Single Plex Assays

To Luminex

Multiplex

Assays



76






0

500

1000

1500

2000

2500

3000

3500

4000


Sample

Sig

nal In

ten

sit

y (

MF

I)

let-7c

Let-7a

77


miR-107 vs miR-103

0

1000

2000

3000

4000

5000

6000


Sample

Sig

na

l In

ten

sit

y (

MF

I)

miR-103

miR-107









0

1000

2000

3000

4000

5000

6000

7000

8000

9000

10000

hsa-m

iR-1

07

hsa-m

iR-1

47

hsa-m

iR-1

81a

hsa-m

iR-1

92

hsa-m

iR-1

9b

hsa-m

iR-2

20a

hsa-m

iR-2

1hsa-m

iR-2

18

hsa-le

t-7c

hsa-m

iR-1

00

hsa-m

iR-1

25b

hsa-m

iR-

hsa-m

iR-2

05

hsa-m

iR-2

21

hsa-m

iR-2

00a

hsa-m

iR-2

00c

hsa-m

iR-2

06

hsa-m

iR-1

0a

hsa-m

iR-1

6

hsa-m

iR-9

hsa-m

iR-4

24

hsa-m

iR-2

0a

hsa-m

iR-2

0b

hsa-le

t-7a

hsa-m

iR-9

5hsa-m

iR-1

36

hsa-m

iR-1

95

hsa-m

iR-2

10

hsa-m

iR-2

12

hsa-m

iR-

hsa-m

iR-1

7

hsa-m

iR-1

22

hsa-m

iR-1

43

hsa-m

iR-1

45

hsa-m

iR-1

06a

hsa-m

iR-

hsa-m

iR-1

0b

hsa-m

iR-1

hsa-m

iR-2

23

hsa-m

iR-1

81b

hsa-m

iR-1

55

hsa-le

t-7g

hsa-le

t-7i

hsa-m

iR-9

6

hsa-m

iR-1

37

hsa-m

iR-1

85

hsa-m

iR-1

38

hsa-m

iR-1

98

hsa-m

iR-1

33a

hsa-m

iR-1

33b

hsa-m

iR-1

5a

hsa-m

iR-3

4a

hsa-m

iR-2

9a

hsa-m

iR-2

9b

hsa-m

iR-1

03

hsa-m

iR-2

22

hsa-m

iR-1

46a

cel-m

iR-3

9cel-m

iR-5

4

cel-m

iR-2

38

Regions

Ne

t S

ign

al In

ten

sit

y (

MF

I)

106a

17


Total RNA Samples

53-Plex Assay

miR-9 miR-122 miR-1

0

5000

10000

15000

20000

25000

Samples

Ne

t S

ign

al In

ten

sit

y (

MF

I)

miR-9

miR-122

miR-1






289704

12431797

4050

7044

12899

18030

24973

23884

21852

19249

11326

6573

3852

2471

0

5000

10000

15000

20000

25000

30000


Input Amount

Ne

t S

ign

al In

ten

sit

y (

MF

I)

200 amol


81


0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000



Ne

t S

ign

al in

ten

sit

y (

MF

I)

82

miR-133a

000

020

040

060

080

100

120


Total RNA Samples

Rela

tive C

han

ge t

o H

eart

FlexmiR v2

qRT-PCR

Tissue

Re

lati

ve

Ch

an

ge


LNP-miRNA

qRT-PCR

83

miRNA

Re

lati

ve

Ch

an

ge



0

5

10

15

20

25


miRNA

Rela

tive C

han

ge

FlexmiR v2

qRT-PCR

LNP-miRNA

qPCR

84



0

5000

10000

15000

20000

25000

30000

hsa-m

iR-1

07

hsa-m

iR-1

47

hsa-m

iR-1

81a

hsa-m

iR-1

92

hsa-m

iR-1

9b

hsa-m

iR-2

20a

hsa-m

iR-2

1hsa-m

iR-2

18

hsa-le

t-7c

hsa-m

iR-1

00

hsa-m

iR-1

25b

hsa-m

iR-

hsa-m

iR-2

05

hsa-m

iR-2

21

hsa-m

iR-2

00a

hsa-m

iR-2

00c

hsa-m

iR-2

06

hsa-m

iR-1

0a

hsa-m

iR-1

6hsa-m

iR-9

hsa-m

iR-4

24

hsa-m

iR-2

0a

hsa-m

iR-2

0b

hsa-le

t-7a

hsa-m

iR-9

5hsa-m

iR-1

36

hsa-m

iR-1

95

hsa-m

iR-2

10

hsa-m

iR-2

12

hsa-m

iR-

hsa-m

iR-1

7hsa-m

iR-1

22

hsa-m

iR-1

43

hsa-m

iR-1

45

hsa-m

ir-1

06a

hsa-m

iR-

hsa-m

iR-1

0b

hsa-m

iR-1

hsa-m

iR-2

23

hsa-m

iR-1

81b

hsa-le

t-155

hsa-le

t-7g

hsa-m

iR-7

ihsa-m

iR-9

6hsa-m

iR-1

37

hsa-m

iR-1

85

hsa-m

iR-1

38

hsa-m

iR-1

33a

hsa-m

iR-1

33b

hsa-m

iR-1

5a

hsa-m

iR-3

4a

hsa-m

iR-2

9a

hsa-m

iR-2

9b

hsa-m

iR-1

03

hsa-m

iR-2

22

hsa-m

iR-1

46a

hsa-m

iR-3

9cel-m

iR-3

9cel-m

iR-2

38

Regions

Ne

t S

ign

al in

ten

sit

y (

MF

I)

50ng

100ng

250ng

500ng








bull Flexible





Proteomic Assays



Nucleic Acids








or IgM





Multiplex Detection

Detection


IgG

1 2 3 4





















Y Y

Y

IgG1 IgG2

IgG3

IgG4 IgA



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Detection



Antigen

1 2 3








Y Y Y

Y

Y

Y Y

Y Y

Y Y

Y



Y


Detection

Multiplex Detection


















compounds





compounds



SAPE SAPE














SAPE SAPE


Detection

Multiplex Detection












543-550






GTPases 827 271-282














Single Plex Assays

To Luminex

Multiplex

Assays



77


miR-107 vs miR-103

0

1000

2000

3000

4000

5000

6000


Sample

Sig

na

l In

ten

sit

y (

MF

I)

miR-103

miR-107









0

1000

2000

3000

4000

5000

6000

7000

8000

9000

10000

hsa-m

iR-1

07

hsa-m

iR-1

47

hsa-m

iR-1

81a

hsa-m

iR-1

92

hsa-m

iR-1

9b

hsa-m

iR-2

20a

hsa-m

iR-2

1hsa-m

iR-2

18

hsa-le

t-7c

hsa-m

iR-1

00

hsa-m

iR-1

25b

hsa-m

iR-

hsa-m

iR-2

05

hsa-m

iR-2

21

hsa-m

iR-2

00a

hsa-m

iR-2

00c

hsa-m

iR-2

06

hsa-m

iR-1

0a

hsa-m

iR-1

6

hsa-m

iR-9

hsa-m

iR-4

24

hsa-m

iR-2

0a

hsa-m

iR-2

0b

hsa-le

t-7a

hsa-m

iR-9

5hsa-m

iR-1

36

hsa-m

iR-1

95

hsa-m

iR-2

10

hsa-m

iR-2

12

hsa-m

iR-

hsa-m

iR-1

7

hsa-m

iR-1

22

hsa-m

iR-1

43

hsa-m

iR-1

45

hsa-m

iR-1

06a

hsa-m

iR-

hsa-m

iR-1

0b

hsa-m

iR-1

hsa-m

iR-2

23

hsa-m

iR-1

81b

hsa-m

iR-1

55

hsa-le

t-7g

hsa-le

t-7i

hsa-m

iR-9

6

hsa-m

iR-1

37

hsa-m

iR-1

85

hsa-m

iR-1

38

hsa-m

iR-1

98

hsa-m

iR-1

33a

hsa-m

iR-1

33b

hsa-m

iR-1

5a

hsa-m

iR-3

4a

hsa-m

iR-2

9a

hsa-m

iR-2

9b

hsa-m

iR-1

03

hsa-m

iR-2

22

hsa-m

iR-1

46a

cel-m

iR-3

9cel-m

iR-5

4

cel-m

iR-2

38

Regions

Ne

t S

ign

al In

ten

sit

y (

MF

I)

106a

17


Total RNA Samples

53-Plex Assay

miR-9 miR-122 miR-1

0

5000

10000

15000

20000

25000

Samples

Ne

t S

ign

al In

ten

sit

y (

MF

I)

miR-9

miR-122

miR-1






289704

12431797

4050

7044

12899

18030

24973

23884

21852

19249

11326

6573

3852

2471

0

5000

10000

15000

20000

25000

30000


Input Amount

Ne

t S

ign

al In

ten

sit

y (

MF

I)

200 amol


81


0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000



Ne

t S

ign

al in

ten

sit

y (

MF

I)

82

miR-133a

000

020

040

060

080

100

120


Total RNA Samples

Rela

tive C

han

ge t

o H

eart

FlexmiR v2

qRT-PCR

Tissue

Re

lati

ve

Ch

an

ge


LNP-miRNA

qRT-PCR

83

miRNA

Re

lati

ve

Ch

an

ge



0

5

10

15

20

25


miRNA

Rela

tive C

han

ge

FlexmiR v2

qRT-PCR

LNP-miRNA

qPCR

84



0

5000

10000

15000

20000

25000

30000

hsa-m

iR-1

07

hsa-m

iR-1

47

hsa-m

iR-1

81a

hsa-m

iR-1

92

hsa-m

iR-1

9b

hsa-m

iR-2

20a

hsa-m

iR-2

1hsa-m

iR-2

18

hsa-le

t-7c

hsa-m

iR-1

00

hsa-m

iR-1

25b

hsa-m

iR-

hsa-m

iR-2

05

hsa-m

iR-2

21

hsa-m

iR-2

00a

hsa-m

iR-2

00c

hsa-m

iR-2

06

hsa-m

iR-1

0a

hsa-m

iR-1

6hsa-m

iR-9

hsa-m

iR-4

24

hsa-m

iR-2

0a

hsa-m

iR-2

0b

hsa-le

t-7a

hsa-m

iR-9

5hsa-m

iR-1

36

hsa-m

iR-1

95

hsa-m

iR-2

10

hsa-m

iR-2

12

hsa-m

iR-

hsa-m

iR-1

7hsa-m

iR-1

22

hsa-m

iR-1

43

hsa-m

iR-1

45

hsa-m

ir-1

06a

hsa-m

iR-

hsa-m

iR-1

0b

hsa-m

iR-1

hsa-m

iR-2

23

hsa-m

iR-1

81b

hsa-le

t-155

hsa-le

t-7g

hsa-m

iR-7

ihsa-m

iR-9

6hsa-m

iR-1

37

hsa-m

iR-1

85

hsa-m

iR-1

38

hsa-m

iR-1

33a

hsa-m

iR-1

33b

hsa-m

iR-1

5a

hsa-m

iR-3

4a

hsa-m

iR-2

9a

hsa-m

iR-2

9b

hsa-m

iR-1

03

hsa-m

iR-2

22

hsa-m

iR-1

46a

hsa-m

iR-3

9cel-m

iR-3

9cel-m

iR-2

38

Regions

Ne

t S

ign

al in

ten

sit

y (

MF

I)

50ng

100ng

250ng

500ng








bull Flexible





Proteomic Assays



Nucleic Acids








or IgM





Multiplex Detection

Detection


IgG

1 2 3 4





















Y Y

Y

IgG1 IgG2

IgG3

IgG4 IgA



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Detection



Antigen

1 2 3








Y Y Y

Y

Y

Y Y

Y Y

Y Y

Y



Y


Detection

Multiplex Detection


















compounds





compounds



SAPE SAPE














SAPE SAPE


Detection

Multiplex Detection












543-550






GTPases 827 271-282














Single Plex Assays

To Luminex

Multiplex

Assays





0

1000

2000

3000

4000

5000

6000

7000

8000

9000

10000

hsa-m

iR-1

07

hsa-m

iR-1

47

hsa-m

iR-1

81a

hsa-m

iR-1

92

hsa-m

iR-1

9b

hsa-m

iR-2

20a

hsa-m

iR-2

1hsa-m

iR-2

18

hsa-le

t-7c

hsa-m

iR-1

00

hsa-m

iR-1

25b

hsa-m

iR-

hsa-m

iR-2

05

hsa-m

iR-2

21

hsa-m

iR-2

00a

hsa-m

iR-2

00c

hsa-m

iR-2

06

hsa-m

iR-1

0a

hsa-m

iR-1

6

hsa-m

iR-9

hsa-m

iR-4

24

hsa-m

iR-2

0a

hsa-m

iR-2

0b

hsa-le

t-7a

hsa-m

iR-9

5hsa-m

iR-1

36

hsa-m

iR-1

95

hsa-m

iR-2

10

hsa-m

iR-2

12

hsa-m

iR-

hsa-m

iR-1

7

hsa-m

iR-1

22

hsa-m

iR-1

43

hsa-m

iR-1

45

hsa-m

iR-1

06a

hsa-m

iR-

hsa-m

iR-1

0b

hsa-m

iR-1

hsa-m

iR-2

23

hsa-m

iR-1

81b

hsa-m

iR-1

55

hsa-le

t-7g

hsa-le

t-7i

hsa-m

iR-9

6

hsa-m

iR-1

37

hsa-m

iR-1

85

hsa-m

iR-1

38

hsa-m

iR-1

98

hsa-m

iR-1

33a

hsa-m

iR-1

33b

hsa-m

iR-1

5a

hsa-m

iR-3

4a

hsa-m

iR-2

9a

hsa-m

iR-2

9b

hsa-m

iR-1

03

hsa-m

iR-2

22

hsa-m

iR-1

46a

cel-m

iR-3

9cel-m

iR-5

4

cel-m

iR-2

38

Regions

Ne

t S

ign

al In

ten

sit

y (

MF

I)

106a

17


Total RNA Samples

53-Plex Assay

miR-9 miR-122 miR-1

0

5000

10000

15000

20000

25000

Samples

Ne

t S

ign

al In

ten

sit

y (

MF

I)

miR-9

miR-122

miR-1






289704

12431797

4050

7044

12899

18030

24973

23884

21852

19249

11326

6573

3852

2471

0

5000

10000

15000

20000

25000

30000


Input Amount

Ne

t S

ign

al In

ten

sit

y (

MF

I)

200 amol


81


0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000



Ne

t S

ign

al in

ten

sit

y (

MF

I)

82

miR-133a

000

020

040

060

080

100

120


Total RNA Samples

Rela

tive C

han

ge t

o H

eart

FlexmiR v2

qRT-PCR

Tissue

Re

lati

ve

Ch

an

ge


LNP-miRNA

qRT-PCR

83

miRNA

Re

lati

ve

Ch

an

ge



0

5

10

15

20

25


miRNA

Rela

tive C

han

ge

FlexmiR v2

qRT-PCR

LNP-miRNA

qPCR

84



0

5000

10000

15000

20000

25000

30000

hsa-m

iR-1

07

hsa-m

iR-1

47

hsa-m

iR-1

81a

hsa-m

iR-1

92

hsa-m

iR-1

9b

hsa-m

iR-2

20a

hsa-m

iR-2

1hsa-m

iR-2

18

hsa-le

t-7c

hsa-m

iR-1

00

hsa-m

iR-1

25b

hsa-m

iR-

hsa-m

iR-2

05

hsa-m

iR-2

21

hsa-m

iR-2

00a

hsa-m

iR-2

00c

hsa-m

iR-2

06

hsa-m

iR-1

0a

hsa-m

iR-1

6hsa-m

iR-9

hsa-m

iR-4

24

hsa-m

iR-2

0a

hsa-m

iR-2

0b

hsa-le

t-7a

hsa-m

iR-9

5hsa-m

iR-1

36

hsa-m

iR-1

95

hsa-m

iR-2

10

hsa-m

iR-2

12

hsa-m

iR-

hsa-m

iR-1

7hsa-m

iR-1

22

hsa-m

iR-1

43

hsa-m

iR-1

45

hsa-m

ir-1

06a

hsa-m

iR-

hsa-m

iR-1

0b

hsa-m

iR-1

hsa-m

iR-2

23

hsa-m

iR-1

81b

hsa-le

t-155

hsa-le

t-7g

hsa-m

iR-7

ihsa-m

iR-9

6hsa-m

iR-1

37

hsa-m

iR-1

85

hsa-m

iR-1

38

hsa-m

iR-1

33a

hsa-m

iR-1

33b

hsa-m

iR-1

5a

hsa-m

iR-3

4a

hsa-m

iR-2

9a

hsa-m

iR-2

9b

hsa-m

iR-1

03

hsa-m

iR-2

22

hsa-m

iR-1

46a

hsa-m

iR-3

9cel-m

iR-3

9cel-m

iR-2

38

Regions

Ne

t S

ign

al in

ten

sit

y (

MF

I)

50ng

100ng

250ng

500ng








bull Flexible





Proteomic Assays



Nucleic Acids








or IgM





Multiplex Detection

Detection


IgG

1 2 3 4





















Y Y

Y

IgG1 IgG2

IgG3

IgG4 IgA



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Detection



Antigen

1 2 3








Y Y Y

Y

Y

Y Y

Y Y

Y Y

Y



Y


Detection

Multiplex Detection


















compounds





compounds



SAPE SAPE














SAPE SAPE


Detection

Multiplex Detection












543-550






GTPases 827 271-282














Single Plex Assays

To Luminex

Multiplex

Assays



Total RNA Samples

53-Plex Assay

miR-9 miR-122 miR-1

0

5000

10000

15000

20000

25000

Samples

Ne

t S

ign

al In

ten

sit

y (

MF

I)

miR-9

miR-122

miR-1






289704

12431797

4050

7044

12899

18030

24973

23884

21852

19249

11326

6573

3852

2471

0

5000

10000

15000

20000

25000

30000


Input Amount

Ne

t S

ign

al In

ten

sit

y (

MF

I)

200 amol


81


0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000



Ne

t S

ign

al in

ten

sit

y (

MF

I)

82

miR-133a

000

020

040

060

080

100

120


Total RNA Samples

Rela

tive C

han

ge t

o H

eart

FlexmiR v2

qRT-PCR

Tissue

Re

lati

ve

Ch

an

ge


LNP-miRNA

qRT-PCR

83

miRNA

Re

lati

ve

Ch

an

ge



0

5

10

15

20

25


miRNA

Rela

tive C

han

ge

FlexmiR v2

qRT-PCR

LNP-miRNA

qPCR

84



0

5000

10000

15000

20000

25000

30000

hsa-m

iR-1

07

hsa-m

iR-1

47

hsa-m

iR-1

81a

hsa-m

iR-1

92

hsa-m

iR-1

9b

hsa-m

iR-2

20a

hsa-m

iR-2

1hsa-m

iR-2

18

hsa-le

t-7c

hsa-m

iR-1

00

hsa-m

iR-1

25b

hsa-m

iR-

hsa-m

iR-2

05

hsa-m

iR-2

21

hsa-m

iR-2

00a

hsa-m

iR-2

00c

hsa-m

iR-2

06

hsa-m

iR-1

0a

hsa-m

iR-1

6hsa-m

iR-9

hsa-m

iR-4

24

hsa-m

iR-2

0a

hsa-m

iR-2

0b

hsa-le

t-7a

hsa-m

iR-9

5hsa-m

iR-1

36

hsa-m

iR-1

95

hsa-m

iR-2

10

hsa-m

iR-2

12

hsa-m

iR-

hsa-m

iR-1

7hsa-m

iR-1

22

hsa-m

iR-1

43

hsa-m

iR-1

45

hsa-m

ir-1

06a

hsa-m

iR-

hsa-m

iR-1

0b

hsa-m

iR-1

hsa-m

iR-2

23

hsa-m

iR-1

81b

hsa-le

t-155

hsa-le

t-7g

hsa-m

iR-7

ihsa-m

iR-9

6hsa-m

iR-1

37

hsa-m

iR-1

85

hsa-m

iR-1

38

hsa-m

iR-1

33a

hsa-m

iR-1

33b

hsa-m

iR-1

5a

hsa-m

iR-3

4a

hsa-m

iR-2

9a

hsa-m

iR-2

9b

hsa-m

iR-1

03

hsa-m

iR-2

22

hsa-m

iR-1

46a

hsa-m

iR-3

9cel-m

iR-3

9cel-m

iR-2

38

Regions

Ne

t S

ign

al in

ten

sit

y (

MF

I)

50ng

100ng

250ng

500ng








bull Flexible





Proteomic Assays



Nucleic Acids








or IgM





Multiplex Detection

Detection


IgG

1 2 3 4





















Y Y

Y

IgG1 IgG2

IgG3

IgG4 IgA



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Detection



Antigen

1 2 3








Y Y Y

Y

Y

Y Y

Y Y

Y Y

Y



Y


Detection

Multiplex Detection


















compounds





compounds



SAPE SAPE














SAPE SAPE


Detection

Multiplex Detection












543-550






GTPases 827 271-282














Single Plex Assays

To Luminex

Multiplex

Assays





289704

12431797

4050

7044

12899

18030

24973

23884

21852

19249

11326

6573

3852

2471

0

5000

10000

15000

20000

25000

30000


Input Amount

Ne

t S

ign

al In

ten

sit

y (

MF

I)

200 amol


81


0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000



Ne

t S

ign

al in

ten

sit

y (

MF

I)

82

miR-133a

000

020

040

060

080

100

120


Total RNA Samples

Rela

tive C

han

ge t

o H

eart

FlexmiR v2

qRT-PCR

Tissue

Re

lati

ve

Ch

an

ge


LNP-miRNA

qRT-PCR

83

miRNA

Re

lati

ve

Ch

an

ge



0

5

10

15

20

25


miRNA

Rela

tive C

han

ge

FlexmiR v2

qRT-PCR

LNP-miRNA

qPCR

84



0

5000

10000

15000

20000

25000

30000

hsa-m

iR-1

07

hsa-m

iR-1

47

hsa-m

iR-1

81a

hsa-m

iR-1

92

hsa-m

iR-1

9b

hsa-m

iR-2

20a

hsa-m

iR-2

1hsa-m

iR-2

18

hsa-le

t-7c

hsa-m

iR-1

00

hsa-m

iR-1

25b

hsa-m

iR-

hsa-m

iR-2

05

hsa-m

iR-2

21

hsa-m

iR-2

00a

hsa-m

iR-2

00c

hsa-m

iR-2

06

hsa-m

iR-1

0a

hsa-m

iR-1

6hsa-m

iR-9

hsa-m

iR-4

24

hsa-m

iR-2

0a

hsa-m

iR-2

0b

hsa-le

t-7a

hsa-m

iR-9

5hsa-m

iR-1

36

hsa-m

iR-1

95

hsa-m

iR-2

10

hsa-m

iR-2

12

hsa-m

iR-

hsa-m

iR-1

7hsa-m

iR-1

22

hsa-m

iR-1

43

hsa-m

iR-1

45

hsa-m

ir-1

06a

hsa-m

iR-

hsa-m

iR-1

0b

hsa-m

iR-1

hsa-m

iR-2

23

hsa-m

iR-1

81b

hsa-le

t-155

hsa-le

t-7g

hsa-m

iR-7

ihsa-m

iR-9

6hsa-m

iR-1

37

hsa-m

iR-1

85

hsa-m

iR-1

38

hsa-m

iR-1

33a

hsa-m

iR-1

33b

hsa-m

iR-1

5a

hsa-m

iR-3

4a

hsa-m

iR-2

9a

hsa-m

iR-2

9b

hsa-m

iR-1

03

hsa-m

iR-2

22

hsa-m

iR-1

46a

hsa-m

iR-3

9cel-m

iR-3

9cel-m

iR-2

38

Regions

Ne

t S

ign

al in

ten

sit

y (

MF

I)

50ng

100ng

250ng

500ng








bull Flexible





Proteomic Assays



Nucleic Acids








or IgM





Multiplex Detection

Detection


IgG

1 2 3 4





















Y Y

Y

IgG1 IgG2

IgG3

IgG4 IgA



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Detection



Antigen

1 2 3








Y Y Y

Y

Y

Y Y

Y Y

Y Y

Y



Y


Detection

Multiplex Detection


















compounds





compounds



SAPE SAPE














SAPE SAPE


Detection

Multiplex Detection












543-550






GTPases 827 271-282














Single Plex Assays

To Luminex

Multiplex

Assays



81


0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000



Ne

t S

ign

al in

ten

sit

y (

MF

I)

82

miR-133a

000

020

040

060

080

100

120


Total RNA Samples

Rela

tive C

han

ge t

o H

eart

FlexmiR v2

qRT-PCR

Tissue

Re

lati

ve

Ch

an

ge


LNP-miRNA

qRT-PCR

83

miRNA

Re

lati

ve

Ch

an

ge



0

5

10

15

20

25


miRNA

Rela

tive C

han

ge

FlexmiR v2

qRT-PCR

LNP-miRNA

qPCR

84



0

5000

10000

15000

20000

25000

30000

hsa-m

iR-1

07

hsa-m

iR-1

47

hsa-m

iR-1

81a

hsa-m

iR-1

92

hsa-m

iR-1

9b

hsa-m

iR-2

20a

hsa-m

iR-2

1hsa-m

iR-2

18

hsa-le

t-7c

hsa-m

iR-1

00

hsa-m

iR-1

25b

hsa-m

iR-

hsa-m

iR-2

05

hsa-m

iR-2

21

hsa-m

iR-2

00a

hsa-m

iR-2

00c

hsa-m

iR-2

06

hsa-m

iR-1

0a

hsa-m

iR-1

6hsa-m

iR-9

hsa-m

iR-4

24

hsa-m

iR-2

0a

hsa-m

iR-2

0b

hsa-le

t-7a

hsa-m

iR-9

5hsa-m

iR-1

36

hsa-m

iR-1

95

hsa-m

iR-2

10

hsa-m

iR-2

12

hsa-m

iR-

hsa-m

iR-1

7hsa-m

iR-1

22

hsa-m

iR-1

43

hsa-m

iR-1

45

hsa-m

ir-1

06a

hsa-m

iR-

hsa-m

iR-1

0b

hsa-m

iR-1

hsa-m

iR-2

23

hsa-m

iR-1

81b

hsa-le

t-155

hsa-le

t-7g

hsa-m

iR-7

ihsa-m

iR-9

6hsa-m

iR-1

37

hsa-m

iR-1

85

hsa-m

iR-1

38

hsa-m

iR-1

33a

hsa-m

iR-1

33b

hsa-m

iR-1

5a

hsa-m

iR-3

4a

hsa-m

iR-2

9a

hsa-m

iR-2

9b

hsa-m

iR-1

03

hsa-m

iR-2

22

hsa-m

iR-1

46a

hsa-m

iR-3

9cel-m

iR-3

9cel-m

iR-2

38

Regions

Ne

t S

ign

al in

ten

sit

y (

MF

I)

50ng

100ng

250ng

500ng








bull Flexible





Proteomic Assays



Nucleic Acids








or IgM





Multiplex Detection

Detection


IgG

1 2 3 4





















Y Y

Y

IgG1 IgG2

IgG3

IgG4 IgA



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Detection



Antigen

1 2 3








Y Y Y

Y

Y

Y Y

Y Y

Y Y

Y



Y


Detection

Multiplex Detection


















compounds





compounds



SAPE SAPE














SAPE SAPE


Detection

Multiplex Detection












543-550






GTPases 827 271-282














Single Plex Assays

To Luminex

Multiplex

Assays



82

miR-133a

000

020

040

060

080

100

120


Total RNA Samples

Rela

tive C

han

ge t

o H

eart

FlexmiR v2

qRT-PCR

Tissue

Re

lati

ve

Ch

an

ge


LNP-miRNA

qRT-PCR

83

miRNA

Re

lati

ve

Ch

an

ge



0

5

10

15

20

25


miRNA

Rela

tive C

han

ge

FlexmiR v2

qRT-PCR

LNP-miRNA

qPCR

84



0

5000

10000

15000

20000

25000

30000

hsa-m

iR-1

07

hsa-m

iR-1

47

hsa-m

iR-1

81a

hsa-m

iR-1

92

hsa-m

iR-1

9b

hsa-m

iR-2

20a

hsa-m

iR-2

1hsa-m

iR-2

18

hsa-le

t-7c

hsa-m

iR-1

00

hsa-m

iR-1

25b

hsa-m

iR-

hsa-m

iR-2

05

hsa-m

iR-2

21

hsa-m

iR-2

00a

hsa-m

iR-2

00c

hsa-m

iR-2

06

hsa-m

iR-1

0a

hsa-m

iR-1

6hsa-m

iR-9

hsa-m

iR-4

24

hsa-m

iR-2

0a

hsa-m

iR-2

0b

hsa-le

t-7a

hsa-m

iR-9

5hsa-m

iR-1

36

hsa-m

iR-1

95

hsa-m

iR-2

10

hsa-m

iR-2

12

hsa-m

iR-

hsa-m

iR-1

7hsa-m

iR-1

22

hsa-m

iR-1

43

hsa-m

iR-1

45

hsa-m

ir-1

06a

hsa-m

iR-

hsa-m

iR-1

0b

hsa-m

iR-1

hsa-m

iR-2

23

hsa-m

iR-1

81b

hsa-le

t-155

hsa-le

t-7g

hsa-m

iR-7

ihsa-m

iR-9

6hsa-m

iR-1

37

hsa-m

iR-1

85

hsa-m

iR-1

38

hsa-m

iR-1

33a

hsa-m

iR-1

33b

hsa-m

iR-1

5a

hsa-m

iR-3

4a

hsa-m

iR-2

9a

hsa-m

iR-2

9b

hsa-m

iR-1

03

hsa-m

iR-2

22

hsa-m

iR-1

46a

hsa-m

iR-3

9cel-m

iR-3

9cel-m

iR-2

38

Regions

Ne

t S

ign

al in

ten

sit

y (

MF

I)

50ng

100ng

250ng

500ng








bull Flexible





Proteomic Assays



Nucleic Acids








or IgM





Multiplex Detection

Detection


IgG

1 2 3 4





















Y Y

Y

IgG1 IgG2

IgG3

IgG4 IgA



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Detection



Antigen

1 2 3








Y Y Y

Y

Y

Y Y

Y Y

Y Y

Y



Y


Detection

Multiplex Detection


















compounds





compounds



SAPE SAPE














SAPE SAPE


Detection

Multiplex Detection












543-550






GTPases 827 271-282














Single Plex Assays

To Luminex

Multiplex

Assays



83

miRNA

Re

lati

ve

Ch

an

ge



0

5

10

15

20

25


miRNA

Rela

tive C

han

ge

FlexmiR v2

qRT-PCR

LNP-miRNA

qPCR

84



0

5000

10000

15000

20000

25000

30000

hsa-m

iR-1

07

hsa-m

iR-1

47

hsa-m

iR-1

81a

hsa-m

iR-1

92

hsa-m

iR-1

9b

hsa-m

iR-2

20a

hsa-m

iR-2

1hsa-m

iR-2

18

hsa-le

t-7c

hsa-m

iR-1

00

hsa-m

iR-1

25b

hsa-m

iR-

hsa-m

iR-2

05

hsa-m

iR-2

21

hsa-m

iR-2

00a

hsa-m

iR-2

00c

hsa-m

iR-2

06

hsa-m

iR-1

0a

hsa-m

iR-1

6hsa-m

iR-9

hsa-m

iR-4

24

hsa-m

iR-2

0a

hsa-m

iR-2

0b

hsa-le

t-7a

hsa-m

iR-9

5hsa-m

iR-1

36

hsa-m

iR-1

95

hsa-m

iR-2

10

hsa-m

iR-2

12

hsa-m

iR-

hsa-m

iR-1

7hsa-m

iR-1

22

hsa-m

iR-1

43

hsa-m

iR-1

45

hsa-m

ir-1

06a

hsa-m

iR-

hsa-m

iR-1

0b

hsa-m

iR-1

hsa-m

iR-2

23

hsa-m

iR-1

81b

hsa-le

t-155

hsa-le

t-7g

hsa-m

iR-7

ihsa-m

iR-9

6hsa-m

iR-1

37

hsa-m

iR-1

85

hsa-m

iR-1

38

hsa-m

iR-1

33a

hsa-m

iR-1

33b

hsa-m

iR-1

5a

hsa-m

iR-3

4a

hsa-m

iR-2

9a

hsa-m

iR-2

9b

hsa-m

iR-1

03

hsa-m

iR-2

22

hsa-m

iR-1

46a

hsa-m

iR-3

9cel-m

iR-3

9cel-m

iR-2

38

Regions

Ne

t S

ign

al in

ten

sit

y (

MF

I)

50ng

100ng

250ng

500ng








bull Flexible





Proteomic Assays



Nucleic Acids








or IgM





Multiplex Detection

Detection


IgG

1 2 3 4





















Y Y

Y

IgG1 IgG2

IgG3

IgG4 IgA



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Detection



Antigen

1 2 3








Y Y Y

Y

Y

Y Y

Y Y

Y Y

Y



Y


Detection

Multiplex Detection


















compounds





compounds



SAPE SAPE














SAPE SAPE


Detection

Multiplex Detection












543-550






GTPases 827 271-282














Single Plex Assays

To Luminex

Multiplex

Assays



84



0

5000

10000

15000

20000

25000

30000

hsa-m

iR-1

07

hsa-m

iR-1

47

hsa-m

iR-1

81a

hsa-m

iR-1

92

hsa-m

iR-1

9b

hsa-m

iR-2

20a

hsa-m

iR-2

1hsa-m

iR-2

18

hsa-le

t-7c

hsa-m

iR-1

00

hsa-m

iR-1

25b

hsa-m

iR-

hsa-m

iR-2

05

hsa-m

iR-2

21

hsa-m

iR-2

00a

hsa-m

iR-2

00c

hsa-m

iR-2

06

hsa-m

iR-1

0a

hsa-m

iR-1

6hsa-m

iR-9

hsa-m

iR-4

24

hsa-m

iR-2

0a

hsa-m

iR-2

0b

hsa-le

t-7a

hsa-m

iR-9

5hsa-m

iR-1

36

hsa-m

iR-1

95

hsa-m

iR-2

10

hsa-m

iR-2

12

hsa-m

iR-

hsa-m

iR-1

7hsa-m

iR-1

22

hsa-m

iR-1

43

hsa-m

iR-1

45

hsa-m

ir-1

06a

hsa-m

iR-

hsa-m

iR-1

0b

hsa-m

iR-1

hsa-m

iR-2

23

hsa-m

iR-1

81b

hsa-le

t-155

hsa-le

t-7g

hsa-m

iR-7

ihsa-m

iR-9

6hsa-m

iR-1

37

hsa-m

iR-1

85

hsa-m

iR-1

38

hsa-m

iR-1

33a

hsa-m

iR-1

33b

hsa-m

iR-1

5a

hsa-m

iR-3

4a

hsa-m

iR-2

9a

hsa-m

iR-2

9b

hsa-m

iR-1

03

hsa-m

iR-2

22

hsa-m

iR-1

46a

hsa-m

iR-3

9cel-m

iR-3

9cel-m

iR-2

38

Regions

Ne

t S

ign

al in

ten

sit

y (

MF

I)

50ng

100ng

250ng

500ng








bull Flexible





Proteomic Assays



Nucleic Acids








or IgM





Multiplex Detection

Detection


IgG

1 2 3 4





















Y Y

Y

IgG1 IgG2

IgG3

IgG4 IgA



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Detection



Antigen

1 2 3








Y Y Y

Y

Y

Y Y

Y Y

Y Y

Y



Y


Detection

Multiplex Detection


















compounds





compounds



SAPE SAPE














SAPE SAPE


Detection

Multiplex Detection












543-550






GTPases 827 271-282














Single Plex Assays

To Luminex

Multiplex

Assays










bull Flexible





Proteomic Assays



Nucleic Acids








or IgM





Multiplex Detection

Detection


IgG

1 2 3 4





















Y Y

Y

IgG1 IgG2

IgG3

IgG4 IgA



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Detection



Antigen

1 2 3








Y Y Y

Y

Y

Y Y

Y Y

Y Y

Y



Y


Detection

Multiplex Detection


















compounds





compounds



SAPE SAPE














SAPE SAPE


Detection

Multiplex Detection












543-550






GTPases 827 271-282














Single Plex Assays

To Luminex

Multiplex

Assays



Proteomic Assays



Nucleic Acids








or IgM





Multiplex Detection

Detection


IgG

1 2 3 4





















Y Y

Y

IgG1 IgG2

IgG3

IgG4 IgA



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Detection



Antigen

1 2 3








Y Y Y

Y

Y

Y Y

Y Y

Y Y

Y



Y


Detection

Multiplex Detection


















compounds





compounds



SAPE SAPE














SAPE SAPE


Detection

Multiplex Detection












543-550






GTPases 827 271-282














Single Plex Assays

To Luminex

Multiplex

Assays





Nucleic Acids








or IgM





Multiplex Detection

Detection


IgG

1 2 3 4





















Y Y

Y

IgG1 IgG2

IgG3

IgG4 IgA



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Detection



Antigen

1 2 3








Y Y Y

Y

Y

Y Y

Y Y

Y Y

Y



Y


Detection

Multiplex Detection


















compounds





compounds



SAPE SAPE














SAPE SAPE


Detection

Multiplex Detection












543-550






GTPases 827 271-282














Single Plex Assays

To Luminex

Multiplex

Assays








or IgM





Multiplex Detection

Detection


IgG

1 2 3 4





















Y Y

Y

IgG1 IgG2

IgG3

IgG4 IgA



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Detection



Antigen

1 2 3








Y Y Y

Y

Y

Y Y

Y Y

Y Y

Y



Y


Detection

Multiplex Detection


















compounds





compounds



SAPE SAPE














SAPE SAPE


Detection

Multiplex Detection












543-550






GTPases 827 271-282














Single Plex Assays

To Luminex

Multiplex

Assays







or IgM





Multiplex Detection

Detection


IgG

1 2 3 4





















Y Y

Y

IgG1 IgG2

IgG3

IgG4 IgA



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Detection



Antigen

1 2 3








Y Y Y

Y

Y

Y Y

Y Y

Y Y

Y



Y


Detection

Multiplex Detection


















compounds





compounds



SAPE SAPE














SAPE SAPE


Detection

Multiplex Detection












543-550






GTPases 827 271-282














Single Plex Assays

To Luminex

Multiplex

Assays





or IgM





Multiplex Detection

Detection


IgG

1 2 3 4





















Y Y

Y

IgG1 IgG2

IgG3

IgG4 IgA



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Detection



Antigen

1 2 3








Y Y Y

Y

Y

Y Y

Y Y

Y Y

Y



Y


Detection

Multiplex Detection


















compounds





compounds



SAPE SAPE














SAPE SAPE


Detection

Multiplex Detection












543-550






GTPases 827 271-282














Single Plex Assays

To Luminex

Multiplex

Assays







Multiplex Detection

Detection


IgG

1 2 3 4





















Y Y

Y

IgG1 IgG2

IgG3

IgG4 IgA



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Detection



Antigen

1 2 3








Y Y Y

Y

Y

Y Y

Y Y

Y Y

Y



Y


Detection

Multiplex Detection


















compounds





compounds



SAPE SAPE














SAPE SAPE


Detection

Multiplex Detection












543-550






GTPases 827 271-282














Single Plex Assays

To Luminex

Multiplex

Assays




Multiplex Detection

Detection


IgG

1 2 3 4





















Y Y

Y

IgG1 IgG2

IgG3

IgG4 IgA



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Detection



Antigen

1 2 3








Y Y Y

Y

Y

Y Y

Y Y

Y Y

Y



Y


Detection

Multiplex Detection


















compounds





compounds



SAPE SAPE














SAPE SAPE


Detection

Multiplex Detection












543-550






GTPases 827 271-282














Single Plex Assays

To Luminex

Multiplex

Assays























Y Y

Y

IgG1 IgG2

IgG3

IgG4 IgA



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Detection



Antigen

1 2 3








Y Y Y

Y

Y

Y Y

Y Y

Y Y

Y



Y


Detection

Multiplex Detection


















compounds





compounds



SAPE SAPE














SAPE SAPE


Detection

Multiplex Detection












543-550






GTPases 827 271-282














Single Plex Assays

To Luminex

Multiplex

Assays





Y Y

Y

IgG1 IgG2

IgG3

IgG4 IgA



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Detection



Antigen

1 2 3








Y Y Y

Y

Y

Y Y

Y Y

Y Y

Y



Y


Detection

Multiplex Detection


















compounds





compounds



SAPE SAPE














SAPE SAPE


Detection

Multiplex Detection












543-550






GTPases 827 271-282














Single Plex Assays

To Luminex

Multiplex

Assays





Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Detection



Antigen

1 2 3








Y Y Y

Y

Y

Y Y

Y Y

Y Y

Y



Y


Detection

Multiplex Detection


















compounds





compounds



SAPE SAPE














SAPE SAPE


Detection

Multiplex Detection












543-550






GTPases 827 271-282














Single Plex Assays

To Luminex

Multiplex

Assays





Y Y

Y



Y Y

Y



Y Y

Y



Y Y

Y



Detection



Antigen

1 2 3








Y Y Y

Y

Y

Y Y

Y Y

Y Y

Y



Y


Detection

Multiplex Detection


















compounds





compounds



SAPE SAPE














SAPE SAPE


Detection

Multiplex Detection












543-550






GTPases 827 271-282














Single Plex Assays

To Luminex

Multiplex

Assays





Y Y

Y



Y Y

Y



Y Y

Y



Detection



Antigen

1 2 3








Y Y Y

Y

Y

Y Y

Y Y

Y Y

Y



Y


Detection

Multiplex Detection


















compounds





compounds



SAPE SAPE














SAPE SAPE


Detection

Multiplex Detection












543-550






GTPases 827 271-282














Single Plex Assays

To Luminex

Multiplex

Assays





Y Y

Y



Y Y

Y



Detection



Antigen

1 2 3








Y Y Y

Y

Y

Y Y

Y Y

Y Y

Y



Y


Detection

Multiplex Detection


















compounds





compounds



SAPE SAPE














SAPE SAPE


Detection

Multiplex Detection












543-550






GTPases 827 271-282














Single Plex Assays

To Luminex

Multiplex

Assays





Y Y

Y



Detection



Antigen

1 2 3








Y Y Y

Y

Y

Y Y

Y Y

Y Y

Y



Y


Detection

Multiplex Detection


















compounds





compounds



SAPE SAPE














SAPE SAPE


Detection

Multiplex Detection












543-550






GTPases 827 271-282














Single Plex Assays

To Luminex

Multiplex

Assays





Detection



Antigen

1 2 3








Y Y Y

Y

Y

Y Y

Y Y

Y Y

Y



Y


Detection

Multiplex Detection


















compounds





compounds



SAPE SAPE














SAPE SAPE


Detection

Multiplex Detection












543-550






GTPases 827 271-282














Single Plex Assays

To Luminex

Multiplex

Assays










Y Y Y

Y

Y

Y Y

Y Y

Y Y

Y



Y


Detection

Multiplex Detection


















compounds





compounds



SAPE SAPE














SAPE SAPE


Detection

Multiplex Detection












543-550






GTPases 827 271-282














Single Plex Assays

To Luminex

Multiplex

Assays









Y Y Y

Y

Y

Y Y

Y Y

Y Y

Y



Y


Detection

Multiplex Detection


















compounds





compounds



SAPE SAPE














SAPE SAPE


Detection

Multiplex Detection












543-550






GTPases 827 271-282














Single Plex Assays

To Luminex

Multiplex

Assays







Y Y Y

Y

Y

Y Y

Y Y

Y Y

Y



Y


Detection

Multiplex Detection


















compounds





compounds



SAPE SAPE














SAPE SAPE


Detection

Multiplex Detection












543-550






GTPases 827 271-282














Single Plex Assays

To Luminex

Multiplex

Assays





Y Y Y

Y

Y

Y Y

Y Y

Y Y

Y



Y


Detection

Multiplex Detection


















compounds





compounds



SAPE SAPE














SAPE SAPE


Detection

Multiplex Detection












543-550






GTPases 827 271-282














Single Plex Assays

To Luminex

Multiplex

Assays





Y


Detection

Multiplex Detection


















compounds





compounds



SAPE SAPE














SAPE SAPE


Detection

Multiplex Detection












543-550






GTPases 827 271-282














Single Plex Assays

To Luminex

Multiplex

Assays




Detection

Multiplex Detection


















compounds





compounds



SAPE SAPE














SAPE SAPE


Detection

Multiplex Detection












543-550






GTPases 827 271-282














Single Plex Assays

To Luminex

Multiplex

Assays



















compounds





compounds



SAPE SAPE














SAPE SAPE


Detection

Multiplex Detection












543-550






GTPases 827 271-282














Single Plex Assays

To Luminex

Multiplex

Assays











compounds





compounds



SAPE SAPE














SAPE SAPE


Detection

Multiplex Detection












543-550






GTPases 827 271-282














Single Plex Assays

To Luminex

Multiplex

Assays










compounds





compounds



SAPE SAPE














SAPE SAPE


Detection

Multiplex Detection












543-550






GTPases 827 271-282














Single Plex Assays

To Luminex

Multiplex

Assays







compounds





compounds



SAPE SAPE














SAPE SAPE


Detection

Multiplex Detection












543-550






GTPases 827 271-282














Single Plex Assays

To Luminex

Multiplex

Assays







compounds



SAPE SAPE














SAPE SAPE


Detection

Multiplex Detection












543-550






GTPases 827 271-282














Single Plex Assays

To Luminex

Multiplex

Assays





SAPE SAPE














SAPE SAPE


Detection

Multiplex Detection












543-550






GTPases 827 271-282














Single Plex Assays

To Luminex

Multiplex

Assays
















SAPE SAPE


Detection

Multiplex Detection












543-550






GTPases 827 271-282














Single Plex Assays

To Luminex

Multiplex

Assays













SAPE SAPE


Detection

Multiplex Detection












543-550






GTPases 827 271-282














Single Plex Assays

To Luminex

Multiplex

Assays











SAPE SAPE


Detection

Multiplex Detection












543-550






GTPases 827 271-282














Single Plex Assays

To Luminex

Multiplex

Assays









SAPE SAPE


Detection

Multiplex Detection












543-550






GTPases 827 271-282














Single Plex Assays

To Luminex

Multiplex

Assays







SAPE SAPE


Detection

Multiplex Detection












543-550






GTPases 827 271-282














Single Plex Assays

To Luminex

Multiplex

Assays





SAPE SAPE


Detection

Multiplex Detection












543-550






GTPases 827 271-282














Single Plex Assays

To Luminex

Multiplex

Assays




Detection

Multiplex Detection












543-550






GTPases 827 271-282














Single Plex Assays

To Luminex

Multiplex

Assays













543-550






GTPases 827 271-282














Single Plex Assays

To Luminex

Multiplex

Assays
















Single Plex Assays

To Luminex

Multiplex

Assays















Single Plex Assays

To Luminex

Multiplex

Assays










Single Plex Assays

To Luminex

Multiplex

Assays







Single Plex Assays

To Luminex

Multiplex

Assays




Single Plex Assays

To Luminex

Multiplex

Assays



Documents

Luminex Based Genetic and Protein Assay Development Presentation