Sh rna 2013

Sample & Assay Technologies- 1 -

SureSilencing™ shRNA Plasmids

Knock Down Your Favorite Genes with Ease and Confidence with shRNA

Wei Cao, [email protected]

.Technical Support:

.Tel: 1-888-503-3187

.Email: [email protected]

.International customers: [email protected]

.Webinar related questions: [email protected]


Topics will be Covered

RNAi Introduction and Challenges1

2 Solutions: SureSilencing shRNA Plasmid

Key Points to Ensure Successful RNAi3

Application Examples 4

Topic II (Today):

Topic I:RNAi High Throughput Screening Applications, Challenges and Solutions; May 6 , 1pm Eastern Time

https://www2.gotomeeting.com/register/786104298


RNA Interference Introduction

What are siRNAs?siRNAs are 21–23nt (nucleotide) dsRNA duplexes with symmetric 2–3nt 3' overhangs and 5'-phosphate and 3'-hydroxyl groups

How does it work?Dicer delivers the siRNAs to a group of proteins called the RISC (RNA-Inducing Silencing Complex)

siRNA duplex unwinds

Once unwound, the single-stranded antisense strand guides RISC to mRNA that has a complementary sequence


RNAi Strategy: siRNA & shRNA

siRNA shRNAOrigin Synthetic oligonucleotides Plasmids or vector basedBiogenesis Introduced into cell by

transfectionSynthesized in cell; enters RNAi pathway

Gene silencing effect

Transient Non-transient, long term effect

Effect on protein production

Decrease protein levels Decrease protein levels

Applications

Quick transient knockdown, not for long term knocking down; not for enrichment

Transient or stable transfection;Build stable cell lines for renewable source of gene knockdown;Transfer shRNA between different vectors;Inheritable silencing.

� The choice of which one to use depends on the question under investigation, the factors such as cell type, time demand, and the need for transient or stable knockdown.

QIAGEN’s Solution

FlexiTube siRNAFlexiPlate siRNA

SureSilencing shRNA Plasmids


Why Knockdown the Expression of a Gene?

RNAi

Target Identification and Validation

Pathway Interrogation

Biomarker and Drug Target Discovery

Gene Function Studies


RNAi Challenges

� RNAi Knockdown Effectiveness

� Differences exist between: Knockdown efficiencies advertised by

companies & observed by researchers

� RNAi Specificity, Off-Target effect (OTE)

1. Sequence-specific OTEs

� Mismatches between the siRNA guide strand and the complementary target

mRNA sequence, ‘seed region’

� siRNAs function like microRNAs

2. Non-sequence-specific OTEs

� Lipid-mediated response - cellular response to RNAi toxicity

� Immune responses to RNAi, such as induction of Interferon pathway

� RISC-dependent off-target effects


Solutions @ QIAGEN




� Available for every gene in the Human, Mouse, and Rat genome

� Guaranteed >70% Knockdown by at least 2 different shRNAs

� Power discovered by thousands of scientists worldwide

� More than 200 publications in a broad range of research fields

� Cancer� Stem Cell � Immunology � Neuroscience � Signal Transduction� Cell Differentiation � Cardiovascular Disease� Infectious Diseases (HIV, HCV)

�Check website: http://www.sabiosciences.com/support_publication.php

�Customer’s success story: http://www.sabiosciences.com/RNAipublication.php


SureSilencing shRNA – How it works

� A vector is introduced into cells and utilizes the U1 promoter to ensure that the shRNA is always expressed

� Dicer cleaves the shRNAinto siRNA.

� The siRNA gene silencing mechanism is followed.


SureSilencing shRNA Plasmids – Mechanism

U1 promoter transcribesa moderate amount of shRNA

The ampicillin resistancemarker and bacterial origin of replication permit amplification for alifetime supply of plasmid

Enrich or Select: 4 Markers: GFP, Nyomycin, Hygromycin, and PuromycinMultiple Designs: 4 Designs for each gene – each sequence targets different region

FACS enrichment Antibiotic-resistance markers: Stable cell line dev elopment

GFP Hyg PuroNeo


The Best shRNA Design Algorithm

Download White Paper “Did Your RNAi Experiment Work?!”http://www.sabiosciences.com/validaternai.pdf


The Best RNAi Design Algorithm

� Ensure Efficacy: Filter many chemical & sequence properties of siRNA known to be important for activity

� Length, between 19bps ~ 30bps

� GC Content, between 32%~55%� Thermostability bias at 5’-end of antisense strand� Avoid tandem repeats and palindromes:

• no internal repeated sequences of length >=4;

• no GC stretch of length >=8;• no repeats of AAA, UUU, GGG or CCC; • no internal palindrome sequences of length >=5;

� Ensure Specificity with Smith-Waterman sequence alignment algorithm, “Better than BLAST”

� Experimentally Validated shRNA Plasmids� 2 of 4 successful designs per gene IS an Enforceabl e Guarantee!

Zhou H, Zeng X, Wang Y and Seyfarth BR. A Three-Phase Algorithm for Computer Aided siRNA Design.

Informatica.2006 30:357-364.


Validation of SureSilencing shRNAs

� Experimental Validation of shRNA Plasmids

� 329 Designs tested, 221 are successful: 67.2 % (>2/3);

� 86 Genes tested, 74 are successful: 86.0 % (>4/5);

� Original publication by The RNAi Consortium (TRC) reports only 31-38 % (~1/3) success rate using the same definition of success

Designs Tested

Successful Designs

Success Rate (%)

Genes Tested

Successful Genes

Success Rate (%)

SABio’s Set 329 221 67.2 86 74 86.0

The RNAi Consortium

2561, 5422 971, 172 381, 312 53 40 75.5

1. RootRoot DE, Hacohen N, Hahn WC, Lander ES, Sabatini DM. “Genome-scale loss-of-function screening with a lentiviral

RNAi library.” Nat Methods. 2006 Sep;3(9):715-9.

2. Moffat J, Grueneberg DA, Yang X, et. al. “A lentiviral RNAi library for human and mouse genes applied to an arrayed viral

high-content screen.” Cell. 2006 Mar 24;124(6):1283-98.


SureSilencing shRNA plasmids- benefits

� Guaranteed >70% gene knockdown efficiency

� Control Off-target effects by multiple shRNA plasmids� Offer 4 shRNA plasmid designs for each gene� Experimentally validated shRNA design algorithm

� Track/ Enrich or Select� Use Neomycin, Puromycin and Hygromycin markers to build stable cell lines and

study long term effects of gene suppression.� Use GFP Marker to track and enrich transfected cells and study short term effects of

gene suppression.

� Convenient and Cost-Effective� Use standard plasmid-based and lipid-mediated transfection methods � Plasmids provide a renewable source of RNA Interference.

� Accepted by thousands of scientists in various rese arch fields


� Search by Gene

� Search by pathway

or disease

SureSilencing shRNA plasmids – search portal

Genome-wide collection of human, mouse and rat gene s.http://sabiosciences.com/shRNA.php


SureSilencing shRNA plasmids – search portal

� Search by pathway or diseases


SureSilencing shRNA plasmids - contents

� Kit Contents:• 4 SureSilencing shRNA Plasmids - transformation grade• One Negative Control - a scrambled artificial sequence• Sequences are provided

� Additional Material Required:

• Transformation: Competent E. coli cells & other reagents for transformation (LB, ampicillin, plates)

• Plasmid Purification: Plasmid purification kit, such as EndoFree Plasmid Maxi Kit (QIAGEN Cat# 12362) and QIAfilter Plasmid Midi Kit (QIAGEN Cat# 12243)

• Transfection: Lipid-mediated transfection reagent (Attractene QIAGEN Cat#301004, or others) or electroporator; Antibiotics: Hygromycin, G418 (for Neomycin), or Puromycin

• Real-time PCR Verification of knockdown: • cDNA synthesis kit (Cat# 330401)• RT2 SYBR Green Master Mix (Cat# 330500)• RT2 Primer Assays – target gene of interest and a housekeeping gene


Workflow for shRNA plasmids knockdown

Start with shRNA Plasmids

3 Steps

1

23

24 or 48hr Incubation

GFP enrich < 1 day

Select with antibiotic ~1-2 week


Workflow for shRNA plasmids knockdown

Assay gene knockdown effect� mRNA level

• Real time qPCR, Northern blot, end-point PCR

� Protein level• SDS-PAGE and Western Verification

• Other Biochemical Assays • Timing is critical (when do I look, when do I do experiment)

RT2 PCR Validation of gene knockdown at mRNA level• cDNA synthesis kit, RT2 SYBR Green Master Mix

• Primers for target gene and housekeeping (control) gene such as ACTB and GAPD

• Template from transfected cells with target gene shRNA and negative control shRNA

• Perform PCR reaction

• Data Analysis


Control shRNATarget shRNA

Target Gene

Control shRNA Target shRNA

Housekeeping Gene

Real-Time PCR Validation of shRNA Knockdown

� Target gene expression decreases by 2 threshold cycles, indicating >70% knockdown;

� Housekeeping gene expression is not altered upon transfection with the target gene shRNA plasmid relative to the control shRNA.


shRNA experiment optimization

� Controls

� Enrichment

� Selection

� Validation

Key points to ensure successful RNAi


Optimization #1 - controls

� Untreated cells: Use normal cells in a normal culture condition as a pure background

� Mock control: For transfection reagent (transient experiment only). Cells treated with transfection reagent only without any shRNA plasmid DNA. Help to identify any effect directly from the transfection reagent

� Non-targeting shRNA control: Use the same shRNA expression vector that will activate RISC and the RNAi pathway, but does not target any human, mouse or rat genes. This allows for examination of the effects of shRNA transfection and RNAi activation on gene expression. Cells transfected with the non-target shRNA vector will also provide a useful reference for interpretation of knockdown.

This negative control is provided with each shRNA p lasmid set


Percent Knockdown

Pre-Sorted Population (%)Knockdown

Sorted Population(%) Knockdown

PRKCAProtein Kinase C alpha

37 71.8 (69.7, 73.8)

TP53Tumor protein p53

52 70.8 (68.4, 73.0)

FACS-based enrichment for GFP-expressing cells

� Transient transfection may have lower efficiency in some cell lines; � Unsorted cells may exhibit lower knockdown due to a large population of untransfected

cells; � Sorting will remove the untransfected cells and enrich the population, thus providing a

true measurement of knockdown;� Peak Ex. of the GFP at 505nm, with a shoulder at 480nm; Em. occurs at 515nm

Optimization #2 - enrichment


Potential challenge when using pooled population� Initial stably antibiotic selected whole pool population appears to

fail, due to: -Random integration sites affect shRNA expression and knockdown efficiency-Average knockdown efficiency of all integration sites is seen, some better than others

� Strategy: Clone stably transfected cells with two best designs, then select by limiting dilution- Leading a high success rate: 2 out of 5 tested now successful

Optimization #3 - selection

Antibiotic selection strategy � Before transfection, determine “effective concentration” of

antibiotics-depends on cell line, growth rate, state of confluence

� After transfection, re-plate cells at a low cell density (≤10%) and grow cells in medium containing the effective concentration of antibiotic

Selection using entire pool

Individual Stably Selected Clones


Optimization #4 - validation

To ensure >70% knockdown efficiency:• Transfection Efficiency (TE): >=80%• Biological Sample Consistency: 3

Validation – qPCR at the mRNA level• PCR Reproducibility• PCR Amplification Efficiency• Site-specific primer may be

necessary for some genes

Validation – at protein levelAt protein level, knockdown is not always immediately apparent

• Need to optimize timing• Protein level measurement – Western blot, enzyme activity assay, reporter assay, etc.

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A B C D E

Designs

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default primer

site specific primer


Application Examples – Case Studies

shRNA Case Studies


shRNA Case Study 1

� Validation of STAT3 RNAi Design in-house

Target GOI (Gene of interest): STAT3 (Signal transducer and activator of transcription 3)

Assay method: Real-time RT-PCR

Cell model: 293H cells

Control HKG (Housekeeping gene): ACTB (Beta actin)


shRNA Case Study #1– Experimental Workflow

shRN

A1

shRN

A3

shRN

A2

shRN

A4

TransfectionGrade shRNAs

Cell Culture:293H cells were cultured in D-MEM with 10% FBS and 1x non-essential amino acids for no more than 15 passages.

shRNA Delivery:Mix 4 STAT3 shRNA plasmids (GFP) (0.8mg) with 3mL Lipofectamine 2000 reagent in a 24-well plate; Change culture media after 24hr transfection.

Check transfection efficiency by GFP expression using fluorescence microscopy

Isolate total RNA:Isolated total RNA after 48 hrs

3 x 4 GOI+ 3 x 1NC = 15

� Validation of STAT3 RNAi Design

Isolate RNA


shRNA Case Study #1– Experimental Workflow

Reverse Transcription:Synthesized cDNA from total RNA (15 samples)

Primer set and Master Mix cocktail:2 cocktails: GOI and HKGCocktail = cDNA + Master mix + H2O

Set up & perform real-time PCR:3 technical replicatesGOI: 15 x 3 =45HKG: 15 x 3 =45

Analyze data:Free data analysis template performs all the calculation & generate report http://www.sabiosciences.com/rnaidataanalysis.php Analyze data & report

Total RNA->cDNA� Validation of STAT3 RNAi Design

15 samples


shRNA Case Study #1– Data Analysis & Report

DATE11/3/2011

Transfection 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3

STAT3 PCR 1 24.00 24.11 24.08 24.44 24.18 24.18 31.54 22.67 23.09 24.31 24.51 24.26 28.96 23.03 22.90STAT3 PCR 2 24.09 24.30 24.29 24.56 24.31 24.32 31.58 22.78 23.33 24.46 24.55 24.35 28.83 22.94 22.82

STAT3 PCR 3 24.02 24.28 24.19 24.57 24.32 24.28 31.92 22.82 23.28 24.47 24.64 24.29 28.87 23.00 22.83

Average Ct 24.04 24.23 24.19 24.52 24.27 24.26 31.68 22.76 23.23 24.41 24.57 24.30 28.89 22.99 22.85

SD Ct 0.05 0.10 0.11 0.07 0.08 0.07 0.21 0.08 0.13 0.09 0.07 0.05 0.07 0.05 0.04

QC 1 OK OK OK OK OK OK OK OK OK OK OK OK OK OK OKACTB PCR 1 17.65 17.74 17.62 17.79 17.82 17.81 26.38 18.03 18.46 17.85 17.87 17.68 25.05 19.18 19.15

ACTB PCR 2 17.83 17.96 17.70 17.86 17.87 17.74 26.39 17.93 18.50 17.86 17.94 17.81 24.90 19.10 19.12ACTB PCR 3 17.72 17.81 17.66 17.77 17.75 17.83 26.45 18.00 18.32 17.69 17.84 17.63 24.97 19.15 19.16

Average Ct 17.73 17.84 17.66 17.81 17.81 17.79 26.41 17.99 18.43 17.80 17.88 17.71 24.97 19.14 19.14

SD Ct 0.09 0.11 0.04 0.05 0.06 0.05 0.04 0.05 0.09 0.10 0.05 0.09 0.08 0.04 0.02

QC 2 OK OK OK OK OK OK OK OK OK OK OK OK OK OK OK

∆Ct 6.30 6.39 6.53 6.72 6.46 6.47 5.27 4.77 4.81 6.61 6.68 6.59 3.91 3.85 3.71

SD ∆Ct 0.10 0.15 0.11 0.09 0.10 0.09 0.21 0.09 0.16 0.13 0.08 0.10 0.10 0.06 0.05

Average ∆Ct

SD ∆Ct BIO

Overall Mean SD ∆Ct

∆∆Ct

Overall SD ∆∆Ct

QC 3Percent of Control

Percent Knock Down+ SD- SD

Report:STAT3-1 ( 80.88 85.48 )STAT3-2 ( 82.39 87.00 )STAT3-3 ( 41.33 64.31 )STAT3-4 ( 84.09 87.18 )

0.28

3.82

0.11

0.11

2.59

0.18

6.55

0.16

0.15

2.72

0.19

6.41

0.13

0.11

OK

1.13

0.32

6.63

0.08

0.05

2.81

0.14

4.95

0.30

1.30

1.43

83.341.91

2.15

84.871.90

2.17

STAT3-4 Negative ControlReal-Time PCR Result for STAT3 shRNA

54.24 85.720.17 0.15 0.46 0.14

OK

95 % Confidence Interval83.3484.87

STAT3-1 STAT3-2 STAT3-3

9.02

11.24

OK OK

54.2485.72

DesignSuccessfulSuccessfulMediocre

Successful

Percent Knock Down

GOI- STAT3

HKG - ACTB


shRNA Case Study #2

� Case Study 2 – Published by customers


shRNA Case Study #2 – Cancer Research

� Study: The role of PELP1/MNAR signaling in Ovarian Tumorig enesis

PELP1/MNAR (Proline-, glutamic acid–, and leucine–rich protein-1): a NR coregulator

� An example using GFP marker to monitor transfection efficiency and screen the

best design, then using selection marker for long te rm knockdown study.

Method:• Model: OVCAR3 cells expressing PELP1/MNAR-shRNA; • Initially used transient transfection assay to screen 4 shRNA plasmids with GFP, and

80-90% transfection efficiency was monitored by GFP expression after 24 hrs;• Transfected OVCAR3 cells with 5 ug negative control shRNA or 2 PELP1/MNAR-

shRNA plasmids with Neomysin;• Selected transfected cells using G418 (1mg/ml) for long term knockdown;• Assayed the knockdown effect of PELP1/MNAR using Western blot after 72 hrs


shRNA Case Study 2 – Cancer Research

• PELP1 expression is ~100% blocked in OVCAR3 by Western blotting; PR, cyclin D1 were down-regulated;• The colonies of PELP1/MNAR/shRNA decreased analyzed by soft agar colony formation assay;• The expression of Src, AKT and MAPK were decreased by down-regulation of PELP1, by Western analysis of total protein lysates with phospho-specific antibodies

Conclusion: PELP1/MNAR plays a critical role in the proliferation of ovarian cancer cells.

OVCAR3OVCAR3

~100% knockdown of PELP1 expression in OVCAR3 cells


SureSilencing shRNA Plasmids - Benefits

� Efficiency and Specificity: Guaranteed success (> 70% gene knockdown by 2 different shRNA plasmids) & Minimized off-target effects

� Flexibility: 4 markers, GFP, Neomycin, Hygromycin, Puromycin, allow for transient and long-term selections

� Convenient & Cost-effective: Use standard plasmid-based and lipid-mediated transfection methods. Plasmids provide a renewable shRNA resource of RNAi

� Genome Wide RNAi tool for Human, Mouse and Rat genes

� Search portal: Easy to search your gene of interest.

Search by gene or by Pathway or Diseasehttp://www.sabiosciences.com/RNAisearch.phphttp://www.Qiagen.com


SureSilencing shRNAs for Every Gene- A complete system for RNAi from QIAGEN

shRNAs Transfection Purification Analysis

Attractene

� High efficiency and low cytotoxicity for DNA transfection

� Suitable for all adherent cells and sensitive cells

� Ideal for co-transfection and vector-based RNAi (shRNA)

� Free of animal-derived components

RNeasy Kit

� Fast and high-quality total RNA in minutes

� Consistent RNA yields from small amounts of starting material

� No phenol/chloroform extraction, no CsCl gradients, no LiCl or ethanol precipitation

qPCR Primer assaySYBR® Green-based

� High performance: bench validated

� Complete genome coverage: human, mouse, rat, rhesus macaque, fly, etc

� Convenience: Within 5-minutes, deliver guaranteed performance


Dicer is required in both the siRNA and miRNA pathways

What’s the phenotypic effect of Dicer knock down on p53 signaling?

Assessing RNA Interference Phenotypes- Cignal Reporter Assay System

Conclusion: The regulation of p53 signaling is tightly controlled by

microRNA and/or siRNA processing.

Cignal Reporter Assay System

P53 Reporter

+ Dicer siRNA

■ Dual-luciferase & GFP format

■ Plasmid based reporter assay

■ Lentivirial based reporter assay


Thank You for Attending!

Call 888-503-3187 to order

Email: [email protected]

Wei Cao: [email protected]

Search Portalhttp://sabioscience.com/RNAi.php

www.qiagen.comwww.GeneGlobe.com

SureSilencing shRNAs for Every Gene- A complete system from QIAGEN

SureSilencing shRNA Plasmids� available for EVERY human, mouse, and rat gene

� per gene set - 4 designs and 1 control� 4 Marker Selection: GFP, Neomycin, Puromycin and Hygromycin

Documents

Sh rna 2013