SureSilencing shRNA Plasmids Knock Down Your Favorite Genes with Ease and Confidence with shRNA

Wei Cao, Ph.D.

Wei.Cao@QIAGEN.com

Technical Support: Tel: 1-888-503-3187 Email: support@SABiosciences.com International customers: sabio@qiagen.com Webinar related questions: qiawebinars@qiagen.com
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Online Seminar @ QIAGEN - A great way to learn new technologies

http://sabioscience.com/seminarlist.php

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Topics will be Covered

Topic I:
RNAi High Throughput Screening Applications, Challenges and Solutions; April 5, 1pm Eastern Time https://www2.gotomeeting.com/register/772647330

Topic II (Today):
1 RNAi Introduction and Challenges

Solutions: SureSilencing shRNA Plasmid

Key Points to Ensure Successful RNAi

Application Examples

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RNA Interference Introduction

What are siRNAs?
siRNAs are 2123nt (nucleotide) dsRNA duplexes with symmetric 23nt 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

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RNAi Strategies: shRNA & siRNA

siRNA
Origin Biogenesis Synthetic oligonucleotides Introduced into cell by transfection Transient

shRNA
Plasmids or vector based Synthesized in cell; enters RNAi pathway Non-transient, long term effect Decrease protein levels Transient or stable transfection; Build stable cell lines for renewable source of gene knockdown; Transfer shRNA between different vectors; Inheritable silencing.

Gene silencing effect Effect on protein Decrease protein levels production Quick transient knockdown, not for long term knocking down; not for enrichment Applications

QIAGENs Solution

Flexitube, Flexiplate siRNA

SureSilencing shRNA Plasmids

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

RNAi

Gene Function Studies

Pathway Interrogation

Target Identification and Validation

Biomarker and Drug Target Discovery

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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

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Solutions @ QIAGEN

SureSilencing shRNA Plasmids

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SureSilencing shRNA Plasmids

Guaranteed >70% Knockdown for Every Human, Mouse, and Rat Gene 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 http://www.sabiosciences.com/RNAipublication.php

Customers success story:

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SureSilencing shRNA How it works

shRNA Plasmid

A vector is introduced into cells and utilizes the U1 promoter to ensure that the shRNA is always expressed. Dicer cleaves the shRNA into siRNA. The siRNA gene silencing mechanism is followed.

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SureSilencing shRNA Plasmids Mechanism

Amp: Propagation
shRNA Plasmid

U1 promoter Expression

Cell Selection
FACS enrichment Antibiotic-resistance markers: Stable Cell Line Development

GFP

Neo

Hyg

Puro

Enrich or Select: 4 Markers: GFP, Nyomycin, Hygromycin, and Puromycin; Multiple Designs: 4 Designs for each gene each sequence targets different region

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The Best shRNA Design Algorithm

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

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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 Enforceable Guarantee!

Zhou H, Zeng X, Wang Y and Seyfarth BR. A Three-Phase Algorithm for Computer Aided siRNA Design. Informatica.2006 30:357-364.
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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 SABios Set The RNAi Consortium 329 2561, 5422

Successful Designs 221 971, 172

Success Rate (%) 67.2 381, 312

Genes Tested 86 53

Successful Genes 74 40

Success Rate (%) 86.0 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.

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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 research fields

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SureSilencing shRNA plasmids search portal

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

Search by Gene Search by pathway or disease

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SureSilencing shRNA plasmids search portal

Search by pathway or diseases

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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

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Workflow for shRNA plasmids knockdown

3 Steps
Start with shRNA Plasmids
GFP enrich < 1 day

1
Select with antibiotic ~1-2 week

3 2

24 or 48 Incubation

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Workflow for shRNA plasmids knockdown

Assay effects of gene knockdown
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

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Real-Time PCR Validation of shRNA Knockdown

Housekeeping Gene
Control shRNA Target shRNA

Target Gene
Control shRNA Target shRNA

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.

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shRNA experiment optimization

Key points to ensure successful RNAi

Controls Enrichment Selection Validation

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Optimization #1 - controls
Untreated cells:
background Use normal cells in a normal culture condition as a pure

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 plasmid set

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Optimization #2 - enrichment
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
Pre-Sorted Population (%) Knockdown 37 Sorted Population(%) Knockdown 71.8 (69.7, 73.8)

Percent Knockdown PRKCA

Protein Kinase C alpha

TP53
Tumor protein p53

52

70.8 (68.4, 73.0)

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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

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 Individual Stably Selected Clones

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

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Optimization #4 - validation
To ensure >70% knockdown efficiency:
Knocking down efficiency (% )

Transfection Efficiency (TE): >=80% Biological Sample Consistency: 3

100 90 80 70 60 50 40 30 20 10 0 A B C Designs D E default primer site specific primer

Validation qPCR at the mRNA level

PCR Reproducibility PCR Amplification Efficiency Site-specific primer may be necessary for some genes

Validation at protein level

At 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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shRNA Case Studies

Application Examples Case Studies

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shRNA Case Study 1

Validation of STAT3 RNAi Design in-house
Target GOI (Gene of interest): STAT3 (Signal transducer and activator of transcription 3) Cell model: 293H cells Assay method: Real-time RT-PCR Control HKG (Housekeeping gene): ACTB (Beta actin)

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shRNA Case Study #1 Experimental Workflow

Validation of STAT3 RNAi Design
Cell Culture: 293H cells were cultured in D-MEM with 10% FBS and 1x non-essential amino acids for no more than 15 passages.
Transfection Grade shRNAs

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

shRN A1

shRN A2

shRN A3

shRN A4

Isolate RNA

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shRNA Case Study #1 Experimental Workflow

Validation of STAT3 RNAi Design
Total RNA->cDNA Reverse Transcription: Synthesized cDNA from total RNA (15 samples)

15 samples

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

Set up & perform real-time PCR: 3 technical replicates GOI: 15 x 3 =45 HKG: 15 x 3 =45 Analyze data: Free data analysis template performs all the calculation & generate report http://www.sabiosciences.com/rnaidataanalysis.php
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Analyze data & report

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shRNA Case Study #1 Data Analysis & Report

DATE 11/3/2011 Transfection STAT3 PCR 1 STAT3 PCR 2 STAT3 PCR 3 Average Ct SD Ct QC 1 ACTB PCR 1 ACTB PCR 2 ACTB PCR 3 Average Ct SD Ct QC 2 Ct SD Ct Average Ct SD Ct BIO Overall Mean SD Ct Ct Overall SD Ct QC 3 Percent of Control Percent Knock Down + SD - SD Report:
STAT3-1 STAT3-2 STAT3-3 STAT3-4

STAT3-1
1 24.00 24.09 24.02 24.04 0.05 OK 17.65 17.83 17.72 17.73 0.09 OK 6.30 0.10 2 24.11 24.30 24.28 24.23 0.10 OK 17.74 17.96 17.81 17.84 0.11 OK 6.39 0.15 6.41 0.11 0.13 2.59 0.18 OK 0.17 83.34 1.91 2.15 3 24.08 24.29 24.19 24.19 0.11 OK 17.62 17.70 17.66 17.66 0.04 OK 6.53 0.11 1 24.44 24.56 24.57 24.52 0.07 OK 17.79 17.86 17.77 17.81 0.05 OK 6.72 0.09

STAT3-2
2 24.18 24.31 24.32 24.27 0.08 OK 17.82 17.87 17.75 17.81 0.06 OK 6.46 0.10 6.55 0.15 0.16 2.72 0.19 OK 0.15 84.87 1.90 2.17

Real-Time PCR Result for STAT3 shRNA STAT3-3 STAT3-4

3 24.18 24.32 24.28 24.26 0.07 OK 17.81 17.74 17.83 17.79 0.05 OK 6.47 0.09 1 31.54 31.58 31.92 31.68 0.21 OK 26.38 26.39 26.45 26.41 0.04 OK 5.27 0.21 2 22.67 22.78 22.82 22.76 0.08 OK 18.03 17.93 18.00 17.99 0.05 OK 4.77 0.09 4.95 0.28 0.30 1.13 0.32 OK 0.46 54.24 9.02 11.24 3 23.09 23.33 23.28 23.23 0.13 OK 18.46 18.50 18.32 18.43 0.09 OK 4.81 0.16 1 24.31 24.46 24.47 24.41 0.09 OK 17.85 17.86 17.69 17.80 0.10 OK 6.61 0.13 2 24.51 24.55 24.64 24.57 0.07 OK 17.87 17.94 17.84 17.88 0.05 OK 6.68 0.08 6.63 0.05 0.08 2.81 0.14 OK 0.14 85.72 1.30 1.43 3 24.26 24.35 24.29 24.30 0.05 OK 17.68 17.81 17.63 17.71 0.09 OK 6.59 0.10

Negative Control
1 28.96 28.83 28.87 28.89 0.07 OK 25.05 24.90 24.97 24.97 0.08 OK 3.91 0.10 2 23.03 22.94 23.00 22.99 0.05 OK 19.18 19.10 19.15 19.14 0.04 OK 3.85 0.06 3.82 0.11 0.11 3 22.90 22.82 22.83 22.85 0.04 OK 19.15 19.12 19.16 19.14 0.02 OK 3.71 0.05

GOI- STAT3

HKG - ACTB

Percent Knock Down

83.34 84.87 54.24 85.72

95 % Confidence Interval
( ( ( ( 80.88 82.39 41.33 84.09 85.48 87.00 64.31 87.18 ) ) ) )

Design
Successful Successful Mediocre Successful

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shRNA Case Study #2

Case Study 2 Published by customers

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shRNA Case Study #2 Cancer Research

Study: The role of PELP1/MNAR signaling in Ovarian Tumorigenesis
PELP1/MNAR (Proline-, glutamic acid, and leucinerich 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 term 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/MNARshRNA 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

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shRNA Case Study 2 Cancer Research

~100% knockdown of PELP1 expression in OVCAR3 cells

OVCAR3 OVCAR3 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.
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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 lipidmediated 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 Disease http://www.sabiosciences.com/RNAisearch.php http://www.Qiagen.com
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SureSilencing shRNAs for Every Gene

- A complete system for RNAi from QIAGEN
shRNAs Transfection

Purification

Analysis

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)

Attractene

Free of animal-derived components

Fast and high-quality total RNA in minutes Consistent RNA yields from small amounts of starting material RNeasy Kit No phenol/chloroform extraction, no CsCl gradients, no LiCl or ethanol precipitation

High performance: bench validated Complete genome coverage: human, mouse, rat, rhesus macaque, fly, etc qPCR Primer assay SYBR Green-based Convenience: Within 5-minutes, deliver guaranteed performance

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Assessing RNA Interference Phenotypes - by Cignal Reporter Assay System

Cignal Reporter Assay System

Dual-luciferase & GFP format Plasmid based reporter assay Lentivirial based reporter assay

Dicer is required in both the siRNA and miRNA pathways Whats the phenotypic effect of Dicer knock down on p53 signaling?
P53 Reporter + Dicer siRNA

Conclusion: The regulation of p53 signaling is tightly controlled by microRNA and/or siRNA processing.
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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

Search Portal
http://sabioscience.com/RNAi.php www.qiagen.com www.GeneGlobe.com

Thank You for Attending!

20% off of your next shRNA or siRNA orders (+ a Qiagen logo digital clock) Promo code: FDK-WN20C12 Call 888-503-3187 to order Email: support@SABioscience.com

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