Cignal webina

1

Cignal™ Reporter Assays

Simple and Robust Signal Transduction Assays

Brian McNally, Ph.D. Associate Product Manager

2

Topics to be Covered

• Who is SABiosciences?

• Utility of the Cignal Reporter Assays• Challenges Associated with Cell-Based Assays• Solutions Cignal Reporter Assays Provide

• How Cignal Reporter Assays Work• Cignal Pathway Reporters Product Family• Dual-Luciferase Assays• Luciferase Engineered for High Performance• Optimized Transcriptional Regulatory Elements (TREs)

• How YOU Can Use Cignal Reporter Assays• Performance and applications data.

3

Who IS SABiosciences?

• Located in Frederick, Maryland, USA (near to NIH Bethesda campus)

• Founded 10 years ago by scientists working at the NIH• Core Values: Innovation, Focus, Systematic Design• Goal: Transform cutting-edge biological information and

technologies into easy to use, accessible, and innovative systems biology research tools.

• Products: Gene expression analysis, protein function analysis and cell based assays

4

Systems Biology @ SABiosciences

DNA

RNA

Protein

SequencingSNP DetectionChIPMethylation

ChIP-qPCR AssaysMethyl Profiler

Discovery Screening

Confirmation

Gene Expression Service CoreRT2 PCR ArraysRT2 miRNA Arrays and AssaysRT2 qPCR Assays

DetectionKnockdownReporter SystemPhenotypes

ELISArray KitssiRNA Arrays / shRNA PlasmidsCignal Reporter SystemCASE Kits

Genomics &Regulation

Gene Expression

Gene FunctionProtein Function

Technologies SABiosciences ProductsResearch

5

Topics to be Covered• Who is SABiosciences?

• Utility of the Cignal Reporter Assays• Challenges Associated with Cell-Based

Assays• Solutions Cignal Reporter Assays Provide

• How Cignal Reporter Assays Work• Cignal Pathway Reporters Product Family• Dual-Luciferase Assays• Luciferase Engineered for High Performance• Optimized Transcriptional Regulatory Elements (TREs)

• How YOU Can Use Cignal Reporter Assays• Performance and applications data.

6

Cignal Applications

• Monitoring signaling pathway activationIs my pathway of interest regulated by biological molecule “X”?

• Studying the biological response of a drug candidate or chemical compound Does my compound of interest regulate pathway “X”?

• Phenotypic analysis of gene knockdown or gene over-expressionDoes my gene of interest regulate pathway “X”?

• Mode of action of protein, peptide and ligandsDoes my protein/peptide/ligand of interest regulate pathway “X”?

7

Cell-based Assay Research Challenges

• Variability within experimentsWell-to-well and plate-to-plate reproducibility are notoriously challenging with cell-based assays.

• Variability between experimentsReliable results often depend upon the “status” of the cells, and who in the lab is conducting the experiments.

• Assay PerformanceSensitivity, specificity, signal to noise ratio, and simplicity are of utmost importance.

8

What Cignal Reporter Assays Provide

• Functionally validated dual-reporter formulation Minimizes variability, increasing the biological relevance of each experiment

• Ready-to-use reporters and controls, coupled with a transient reporter systemEnables simple and rapid analysis of the regulation of 29 signal transduction pathways

• Combination of custom-engineered transcriptional regulatory elements (TREs) and destabilized, codon- optimized firefly luciferaseIncreases the signal to noise ratio and maximizes assay sensitivity and specificity

9

Topics to be Covered

• Who is SABiosciences?• Utility of the Cignal Reporter Assays

• Challenges Associated with Cell-Based Assays• Solutions Cignal Reporter Assays Provide

• How Cignal Reporter Assays Work• Cignal Pathway Reporters Product Family• Dual-Luciferase Assays• Luciferase Engineered for High Performance• Optimized Transcriptional Regulatory Elements (TREs)

• How YOU Can Use Cignal Reporter Assays• Performance and applications data.

10

Cignal Pathway Reporter System Products

Easy totransfect cell lines

Primary cells, stem cells,and difficult to transfect cell lines

EndpointAssays

EndpointAssays

Dynamic Live Cell Assays

Dynamic Live Cell Assays

Cignal Dual-LuciferaseReporter Assays

Cignal LentiLuciferase Reporters

Cignal GFP Reporter Assays

Cignal LentiGFP Reporters

Single PathwayAssays

Single PathwayAssays

Single PathwayAssays

Single PathwayAssays

10-PathwayArrays

10-PathwayArrays

11

Cignal Reporter AssaysPathway Transcription FactorAmino Acid Deprivation ATF4/ATF3/ATF2Androgen Androgen ReceptorAntioxidant Response Nrf2 & Nrf1ATF6 ATF6C/EBP C/EBPcAMP/PKA CREB Cell Cycle E2F/DP1 DNA Damage p53 EGR1 EGR1ER Stress CBF/NF-Y/YY1Estrogen ER GATA GATAGlucocorticoid GRHeat Shock Response HSFHeavy Metal Stress MTF1Hedgehog GLIHypoxia HIF-1 Type I Interferon STAT1/STAT2 Interferon Gamma STAT1/STAT1

Pathway Transcription FactorMAPK/ERK Elk-1/SRF MAPK/JNK AP-1 MEF2 MEF2c-myc Myc/Max NFκB NFκBNotch RBP-JkOct4 Oct4PI3K/AKT FOXOPKC/Ca++ NFAT PPAR PPARRetinoic Acid RAR SP1 SP1STAT3 STAT3TGFβ

SMAD2/3/4 VDRE Vitamin D ReceptorWnt TCF/LEF

12

REPORTER DESIGN: Dual-Luciferase Assays

Reporter Construct Firefly luciferaseTATAboxTRE

Renilla luciferaseTATAboxCMVNormalization Construct

Pathway-targeted transcriptionalregulatory elements (35)

Constitutive transcriptionalregulatory element (1)

The Transcriptional Regulatory Elements (TREs) are what establish the

pathway specificity of each reporter!

13

Using Cignal Reporters: What You Need

• Cignal Reporter Assay

• Reliable transfection reagent and protocol (SureFECT)

• Test Biological• siRNA (SureSilencing siRNA Arrays)• shRNA (SureSilencing shRNA)• expression vectors• protein/peptide• small molecule

• Detection Instrument and Luciferase Substrates

14

REPORTER DESIGN: Dual-Luciferase Assays

Reporter Construct Firefly luciferaseTATAboxTRE

Renilla luciferaseTATAboxCMVNormalization Construct

Pathway-targeted transcriptionalregulatory elements (35)

Constitutive transcriptionalregulatory element (1)

The Transcriptional Regulatory Elements (TREs) are what establish the

pathway specificity of each reporter!

15

Dual Luciferase Assays: Why Is It Important?

Sources of Variability in Cell-based Assays:

• Number of cells seeded per well• Transfection efficiencies within and across plates• Multichannel pipettor inconsistencies• “Edge effects” influencing cell culture• Viability of cells following transfection/treatment• Lysis efficiencies within and across plates• Variability inherent to reporter assay

• Changes due to the designed treatments of cells• RNA interference• Overexpression• Recombinant protein/peptide/growth factor• Small molecule

Dual-Luciferase Assaydesign corrects for these unwanted sources of variability.

16

0

5000000

10000000

15000000

20000000

25000000

30000000

Lum

ines

cenc

e (R

elat

ive

Ligh

t Uni

ts)

0

0.5

1

1.5

2

2.5

Rel

ativ

e Lu

cife

rase

Uni

tsDual Luciferase Format Minimizes Variability

Dual-Luciferase Assays:NFkB Reporter Assay

CV=0.09

CV=0.07

Single-Luciferase Assays:NFkB Reporter Assay

NFkB Reporter+ NFkB siRNA

CV=0.69

CV=0.70

NFkB Reporter+ Neg. control siRNA

NFkB Reporter+ NFkB siRNA

NFkB Reporter+ Neg. control siRNA

17

REPORTER DESIGN: Dual-Luciferase Assays

Reporter Construct Firefly luciferaseTATAboxTRE

Renilla luciferaseTATAboxCMVNormalization Construct

Mammalian codon-optimized AND

Destabilized firefly luciferase

The Transcriptional Regulatory Elements (TREs) are what establish the

pathway specificity of each reporter!

18

Engineered Luciferase and the Signal:Noise Ratio

Modified Luciferase:Background NFkB Reporter Signal

(no treatment post-transfection)

Modified Luciferase:NFkB Reporter Signal

Following TNF

Induction

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

Rel

ativ

e Lu

cife

rase

Uni

ts0

50

100

150

200

250

Fold

Indu

ctio

n

WHY?

NegativeControl

NFkB Reporter:Stable Luc

NFkB Reporter:Destabilized Luc

NegativeControl

NFkB Reporter:Stable Luc

NFkB Reporter:Destabilized Luc

19

REPORTER DESIGN: Dual-Luciferase Assays

Reporter Construct Firefly luciferaseTATAboxTRE

Renilla luciferaseTATAboxCMVNormalization Construct

Pathway-targeted transcriptionalregulatory elements (35)

The Transcriptional Regulatory Elements (TREs) are what establish the

pathway specificity of each reporter!

20

Cignal TRE Maximize Luciferase Signal

Dual-Luciferase AssaysUtilizing Cignal p53 TRE

0

20

40

60

80

100

120

140

160

Rel

ativ

e Lu

cife

rase

Uni

ts

0

0.5

1

1.5

2

2.5

3

3.5

Rel

ativ

e Lu

cife

rase

Uni

ts

p53 Reporter+ DMSO

p53 Reporter+ 1µM Doxorubicin

Dual-Luciferase AssaysUtilizing Common p53 TRE

p53 Reporter+ DMSO

p53 Reporter+ 1µM Doxorubicin

125-Fold2.5-Fold

21

Cignal Reporter Assays: How It Works

22

Cignal Reporter Assays (GFP)Pathway Transcription FactorAmino Acid Deprivation ATF4/ATF3/ATF2Androgen Androgen ReceptorAntioxidant Response Nrf2 & Nrf1ATF6 ATF6C/EBP C/EBPcAMP/PKA CREB Cell Cycle E2F/DP1 DNA Damage p53 EGR1 EGR1ER Stress CBF/NF-Y/YY1Estrogen ER GATA GATAGlucocorticoid GRHeat Shock Response HSFHeavy Metal Stress MTF1Hedgehog GLIHypoxia HIF-1 Type I Interferon STAT1/STAT2 Interferon Gamma STAT1/STAT1

Pathway Transcription FactorMAPK/ERK Elk-1/SRF MAPK/JNK AP-1MEF2 MEF2c-myc Myc/MaxNFκB NFκBNotch RBP-JkOct4 Oct4PI3K/AKT FOXOPKC/Ca++ NFAT PPAR PPARRetinoic Acid RAR SP1 SP1STAT3 STAT3TGFβ

SMAD2/3/4VDRE Vitamin D ReceptorWnt TCF/LEF

23

Cignal Reporter Assays: Two Modalities

Dual-luciferase Format- Quantitative endpoint luminescence assay- Exceptional reproducibility, sensitivity, and signal-to-noise ratio- Average expression from a population of cells

GFP Format- Dynamic live cell assay- Single cell resolution- Readout flexibility

(flow cytometry, fluorescent microscopy, fluorometry)

24

Cignal Finder Arrays: Cancer Array Design

Pathway

Cell CycleDNA DamageHypoxiaMAPK/ERKMAPK/JNKc-MycNFkBNotchTGFβWnt

Transcription Factor

E2F/DP1p53HIFElk-1/SRFAP1Myc/MaxNFkBRBP-JkSMAD2/SMAD3/SMAD4TCF/LEF

25

How Does a Cignal Finder Array Work?

26

Topics to be Covered

• Who is SABiosciences?• Utility of the Cignal Reporter Assays

• Challenges Associated with Cell-Based Assays• Solutions Cignal Reporter Assays Provide

• How Cignal Reporter Assays Work• Cignal Pathway Reporters Product Family• Dual-Luciferase Assays• Luciferase Engineered for High Performance• Optimized Transcriptional Regulatory Elements (TREs)

• How YOU Can Use Cignal Reporter Assays• Performance and applications data

27

Cignal Applications

• Functional Genomics – What’s the phenotype of my gene?Does my gene of interest regulate signaling pathway “X”?

• Functional Proteomics – What’s the phenotype of my protein/peptide?Does my protein, peptide, or growth factor of interest regulate

signaling pathway “X”?

• Drug Screening – What is the mechanism of action of my small molecule drug candidates?Do my drug candidates of interest regulate signaling pathway “X”?

28

0

4

8

12

16

20

Cignal Application: RNA Interference

4

8

12

16

20

Cignal Reporter:

siRNA:

NC NC

NC siRNA

p53 p53

NC siRNAp53 siRNA p53 siRNA

Rel

ativ

e Lu

cife

rase

Uni

ts

29

Cignal Applications

• Functional Genomics – What’s the phenotype of my gene?Does my gene of interest regulate signaling pathway “X”?

• Functional Proteomics – What’s the phenotype of my protein/peptide?Does my protein, peptide, or growth factor of interest regulate

signaling pathway “X”?

• Drug Screening – What is the mechanism of action of my small molecule drug candidates?Do my drug candidates of interest regulate signaling pathway “X”?

30

Cignal Application: Functional Proteomics

Rel

ativ

e Lu

cife

rase

Uni

ts

Cignal Reporter:

Protein:

5

10

15

20

25

30

35

NC NC SMAD SMAD SMAD

None None25 ng/µlhuTGFβ1

2.5 ng/mlhuTGFβ1

25 ng/mlhuTGFβ1

31

Cignal Applications

• Functional Genomics – What’s the phenotype of my gene?Does my gene of interest regulate signaling pathway “X”?

• Functional Proteomics – What’s the phenotype of my protein/peptide?Does my protein, peptide, or growth factor of interest regulate

signaling pathway “X”?

• Drug Screening – What is the mechanism of action of my small molecule drug candidates?Do my drug candidates of interest regulate signaling pathway “X”?

32

Cignal Application: Small Molecule Studies

Rel

ativ

e Lu

cife

rase

Uni

ts

Cignal Reporter: CRE CRE CRE CRE

Small Molecule: None 0.1µM Forskolin

50

100

150

200

250

CRE

1µM Forskolin

5µM Forskolin

10µM Forskolin

33

Cignal Application(GFP): Small Molecule Studies

Treatment of Cignal SRE (GFP) transfectants with 10% serum and 10 ng/ml PMAactivates the MAPK/ERK signaling pathway.

Fluorometry Fluorescent Microscopy

34

Application Study

Question:What is impact of p53 gene expression knock down on the p53 signaling pathway AND other cancer-relevantsignaling pathways?

Approach:Carry out a p53 RNA interference experiment with multiple cancer-relevant Cignal Reporter Assays.

p53 Pathway Interaction Study

35

Post-Genomics Research Challenge

Signal TransductionCascadeA B C D

(~40 proteins)

p53 Signaling

36

Cignal Reporters: Constructing Signaling Networks

37

Post-Genomics Research Challenge: Pathway Interactions?

Wnt Signaling NFkB Signaling

E2F Signaling

MAPK/ERK Signaling

Myc Signaling

MAPK/JNK Signaling

TGFβ Signaling

Notch Signaling

?

?

?

?

?

?

?

?

?Hypoxia Signaling

p53 Signaling

38

Cignal Finder Cancer Array : p53 RNAi Study

1 2 3 4 5 6 7 8 11

1. p53 Cignal Reporter

2. RBP-Jk Cignal Reporter

3. TCF/LEF Cignal Reporter

4. E2F Cignal Reporter

5. AP1 Cignal Reporter

6. SRE Cignal Reporter

7. SMAD Cignal Reporter

8. NFkB Cignal Reporter

9. Myc Cignal Reporter

10. HIF Cignal Reporter

11. Cignal Negative Control

12. Cignal Positive Control

129 10

p53 siRNA

Neg. control siRNA

39

Results: Cancer Array Application Study

00.5

11.5

22.5

33.5

44.5

TCF/L

EFRBP-Jk

p53

SMADpR

B-E2F

NFkB Myc HIF

SRE

AP1

Cignal Pathway Reporter

Fold

Cha

nge

in P

athw

ayA

ctiv

atio

n

Impact of p53 siRNA Treatment

40

Post-Genomics Research Challenge: Pathway Interactions?

MAPK/ERK Signaling

Notch Signaling

Hypoxia Signaling

p53 Signaling

41

Topics to be Covered

• Who is SABiosciences?• Utility of the Cignal Reporter Assays

• Challenges Associated with Cell-Based Assays• Solutions Cignal Reporter Assays Provide

• How Cignal Reporter Assays Work• Cignal Pathway Reporters Product Family• Dual-Luciferase Assays• Luciferase Engineered for High Performance• Optimized Transcriptional Regulatory Elements (TREs)

• How YOU Can Use Cignal Reporter Assays• Performance and applications data

42

Summary: Cignal Reporter Assay System• Breadth of Pathways and Research Applications

•35 Pathway-Focused Reporter Assays•4 Application-Specific Cignal Finder 10-Pathway Arrays

• PerformanceExceptional Sensitivity, Specificity, Signal-to-Noise Ratio, and Reproducibility

• ConvenienceReady-to-use, validated, preformatted transient assays

• Dual Modalities•Dual-luciferase quantitative endpoint assays•GFP dynamic live cell assays with single cell resolution

43

Cignal Reporter Assays

Questions?Contact Technical Support 9 AM – 6 PM Eastern M – F

Telephone: 888-503-3187; Email: support@SABiosciences.com

Simple and Robust Signal Transduction Assays