Si rna 2013

Sample & Assay Technologies- 1 -

A Comprehensive Solution for RNAi

HT RNAi Screening

Application, Challenges and Solutions

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Topics will be covered

Overview of RNAi1

siRNA solutions @QIAGEN

2 RNAi application and challenges

3

4 Application examples


RNA interference: a natural phenomenonDiscovery tool, potential therapeutic

Small RNAs Make Big

Splash !The discovery of RNAi earned its

two lead researchers, Andrew Fire

and Craig Mello, the 2006 Nobel

Prize.


What is RNA interference? - The RNAi Pathways

� RNAi: a research toolRNAi silences a target gene through the specific destruction of that gene‘s messenage RNA (mRNA).

� Post-transcriptional gene regulation

� Initiated by double-stranded RNA (dsRNA), processed by DICER

� Effector is ~ 21-24 bp RNA

� Complementary mRNA is cleaved and degraded

� In mammalian cells, dsRNAs >30 bps trigger a nonspecific interferon response, may cause mRNA degradation in a sequence-independent manner.

� ~21 bps are used

http://sabiosciences.com/pathwaycentral.php


The siRNA and miRNA pathways are related

miRNAPathway

siRNAPathway

siRNA

�� !?

From Meister & Tuschl, 2005


RNAi Application

RNAi as a tool for functional analysis

Overview of RNAi1

siRNA solutions @QIAGEN

2 RNAi applications and challenges

3

4 Application examples


Mohr S, Bakal C, Perrimon N. “Genomic screening with RNAi: results and challenges. “Annu Rev Biochem. 2010;79:37-64

RNAi as a tool for functional analysis

Interrogate cellular signaling pathway-system biology

Decode gene functions - functional genomics

Target identification and validation

Drug discovery and disease therapy(Infectious diseases and cancer)

“Learning by Breaking” - simply removing a gene and looking at the effect

What types of questions can be answered by RNAi scre ens?


The Biological question or hypothesis

- 8 -

• Genome-wide screens: seek to identify all possible regulators of a general biological process: cell proliferation or viral infection and replication, etc.

Seek more focused answers• Regulators of apoptosis pathway

• Regulators of DNA damage pathway

• Regulators of signaling by Jak kinase-STAT transcription factors

• …

• Identify an unknown protein

Decide which method to use:

genome-wide screens or more focused screens


RNAi HTS platform

3 steps

dsRNA library stored in 96-well plates

Transfer to 384

or 96 well platesReady-to-

screen 384-

well plate

transfection

Transfection

Add cells

3-7 days incubation to ensure protein depletion

Transcriptional-Luciferase

reporter assaysProtein modification (e.g.

phospho-specific antibodies)

Automated Microscopy

(GFP, fluorescent dyes or

antibody labeling)


RNAi screening challenges� Specificity of RNAi - off-target effect (OTE) is #1 Challenge, can come from:

• Cross-reactivity is a substantial problem

• Mismatches between siRNA guide strand and target mRNA sequence

• ‘seed region’ siRNAs function like microRNAs

• Lipid-mediated response - cellular response to RNAi toxicity

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

• RISC-dependent off-target effects

� Efficiency of RNAi – different genes are turned off with differing efficiencies

� Off-target effects can occur at the level of protein synthesis

� Some cells are notoriously difficult to transfect, or transfection alter the

physiology of the cells

� Specific to HT RNAi Screen: False positive & false negative results


Critical factors for large scale screening

• Maximize on-target effects, minimize off-targets effects

� Effective and specific siRNA design

� siRNA Delivery - Optimize transfection condition

� Multiple screens in multiple cell types

� Validation is always a key – Speficity must be confirmed with multiple siRNAs, and even multiple species

� Controls – Appropriate negative and positive control experiments


Design - siRNA specificity and potency

High quality of siRNA sources

• Refine the standard parameters to select effective and specific siRNAs

• Avoid potential microRNA binding sites

• Avoid Interferon motif – by modifying siRNA sequences

It is IMPOSSIBLE to rule out off-target risks through design alone.

• Good experimental practice is still the key to managing off-target effects!


Choice of siRNA libraries

� Ready to use, QC’d and stable molecules

� Quickest method to knock down a specific gene

� Effective design rules allow potent silencing

� Fairly efficient transfection into many cell types� Control the amount of siRNA more accurately than vector based method

� High throughput (1000’s/day)

� Ability to label and track siRNA

� Modifications easily incorporated (stability, enhanced transfer, potency, etc.)

siRNA is the choice for large scale functional analy sis


Optimization of transfection condition

� Every cell line is differentParameters need to be optimized for each cell line:

� Cell culture density

� Passage number

� Amount of transfection reagent and siRNAs, Ratio

� Complex incubation time on cells

� Optimal time point of analysis

� Proper controls

Statistical analysis:� Strong S/N ratio and low variation

� Before primary screening, optimize assay conditions for negative and positive siRNA control

� Negative and positive siRNA controls should be included on every plate


Controls for siRNA experiments

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

� Mock control: Cells treated with transfection reagent only without any siRNA DNA. Help to identify any effect directly from the transfection reagent

� Negative siRNA control : Well characterized ‘scrambled’ or ‘non-specific sequence’, the best available way for sequence-independent off-target effects

� Positive siRNA control : Ubiquitously expressed (e.g. lamin, actin, g3pdh)

� Assay-specific positive control : Confirm assay is working (when screening for phenotype)


Validation of Screening Hits

� Validation of hits is critical for minimizing the false positive & false

negative in HT siRNA screens

� Multiple screens in multiple cell types

� Achieve early attrition of potential hits� Multiple independent siRNA in primary screen� Decrease variation between replicates

� Key Issues for Validation� Correlation between phenotype and target gene� Redundancy: Confirms specificity of phenotype by multiple independent siRNAs� Rescue by cDNA lacking targeting sequence (eg 5’ UTR)

Key to success: Validation

There should be a clear correlation between protein depletion and phenotypic severity


Validation: Specificity must be confirmed with multiple siRNAs & multiple cell lines

0 20 40 60 80 100

VEGF B duplex 1

VEGF B duplex 2

VEGF B duplex 3

0 20 40 60 80 100

HeLa S3 (1)HeLa S3 (2)HEK 293Hep G2MCF-7



% Normalized Survival


Validation: Redundancy and Rescue- Commentary in Nature Methods: “The Two Rs”

“… the only ways of adequately addressing sequence-dependent off-target effects within RNAi experiments themselves are the ‘the two Rs’.”

Echeverri et al. (2006), Minimizing the risk of reporting false positives in large-scale RNAi screens. Nat Methods, 3(10):777-9

Solution 1: Redundancy Solution 2: Rescue

Specificity Must be Confirmed with Multiple siRNAs !

.Reintroduce an RNAi-resistant version of the target gene product into depleted cells and show recovery of function


� Secondary assays to refine and characterize a functional gene set

� Use complementary assays to those used in the primary screen

� Pharmacological inhibitors or gene mutants should be used to confirm data

from siRNA mediated knockdown

� Compare phenotypes of relevant genes important in the pathway

� Monitor kinetics via multiple time points

� Database mining and computational analysis

Validation: Secondary analysis

Sonia S. and Anjana R. “RNAi screening: tips and techniques”Nat Immunol 2009 10(8):799-804.


QIAGEN provides world-class RNAi solutions:

� QIAGEN’s siRNA design and validation

� Screening solutions: Flexiplate siRNA

� HiPerfect transfection reagent

3 siRNA Solutions @ QIAGEN


� Phase 1 – the “Tuschl Rules” in 2002,

AA(N)19, moderate G.C, a simple BLAST, 50%

active

� Phase 2 – Asymmetry of GC contents in

“active siRNAs” in 2003/2004; Norvatis, Aza Blanc,

Mol. Cell, V12, 2003; Schwarz, cell, 2003, 75%

active

� Phase 3 – BioPred Si. with Norvatis in

2004/2005; 83% active

� Phase 4 – HP OnGuard siRNA 2006/ 2008

�Latest developments: addressing miRNA

related off-Target effects

�Maximal efficiency + minimal off-

target effectsLim et al. 2005, Lewis et al. 2005, Saxena et al. 2003Schwarz , et. al. Cell, Vol. 115, 2003Hall. et al. Nature Biotechnology July 2005

Evolution of siRNA design @ QIAGEN

0

20

40

60

80

100

120

1 11 21 31 41 51 61 71 81 91

% r

emai

ning

mR

NA

1 10 20 30 40 50 60 70 80 90 100

50%

75%

83%

4 duplexes per target, 25 targets


Reduce miRNA related off-target effects- Seed Region Analysis

3’UTR-Seed Region Analysis

� Seed region� Position 2-7 of miRNA / siRNA sequence

� miRNA binding to mRNA through seed region

� Presence of multiple seed region matches increases likelihood of

off-target effects

siRNAs that bind like miRNA

AAAAA3`UTR

CDS

5’5’5’5’ 3’3’3’3’


HP OnGuard siRNA design algorithm- Highest knockdown efficiency and specificity

HP

OnGuard

siRNA

Design

Neural-network

technologyThe world’s largest

siRNA validation

project: >3,700 siRNA

>2 siRNA/target

Homology analysis

Affymetrix

GeneChip analysis

Up-to-date siRNA

target sequences

Asymmetry

3' UTR/seed

region analysis

SNP avoidance

Interferon motif

avoidance

For more information of HP OnGuard siRNA Design:http://www.qiagen.com/Search.aspx?q=hp%2520onguard&l=a5fdae0d-692e-46fe-

b21f-55cca5e19502&pg=1#&&p=1


A Complete solution of RNAi @ QIAGEN

Integration of every step of gene silencing workflow

Step 1 Step 2 Step 3 Step 4

http://www.qiagen.com/Products/Applications/Gene-Silencing/


RNAi Product Portfolio

FlexiTube siRNA Premix

FlexiTube siRNA

FlexiTube GeneSolution

Low ThroughputRNAi

Medium to High Throughput RNAi

Pre

desi

gned

Cus

tom

synt

hesi

s

FlexiPlate siRNA

HP Custom siRNA

AllStars Control siRNA

Vec

tor

base

d

SureSilencing shPlasmids

shRNA webinar: April 19, 1-2pm EDThttps://www2.gotomeeting.com/register/499882890


High throughput screening: FlexiblePlate- Customizable and economical screening solution

FlexiPlate siRNA

� Custom siRNA sets for customer-specified genes and siRNA controls

� 96 or 384-well format

� Select siRNAs from any human, mouse, rat genes

� Validated siRNA, controls, flexible scales (0.1, 0.25, 1 nmol)

� Fast and easy access through QIAGEN’s GeneGlobe

� Up-to-date: GeneGlobe siRNAs are checked regularly for NCBI database updates


Low throughput & validation: FlexiTube siRNA, GeneSolution and Premix

.FlexiTube siRNA� Flexible scale: 1,5,20 nmol for human and

mouse, 5, 20nmol for rat, lyophilized� Pre-annealed, ready for suspension� Chosen from HP GenomeWide or HP

Validated siRNAs� All sequence included� Up-to-date and easy to find and order

from GeneGlobe.com

.FlexiTube GeneSolution siRNA� Same features as above, except you get

4 pre-selected siRNAs for your gene

.FlexiTube siRNA Premix� siRNAs premixed with transfection

reagent


AllStars RNAi controls

http://www.qiagen.com/Products/Applications/Gene-Silencing/

Negative control Use AllStars Negative Control siRNA� No phenotype in cell-based assays� Lowest off-target profile in GeneChip analysis� Shown to enter RISC

Positive control Use AllStars Hs Cell Death Control siRNA� Transfection and knockdown success visible by light microscopy� Ubiquitous utility in all human cell types� For optimization of transfection efficiency

Use AllStars Mm, Rn Cell Death Control siRNA

Extensively characterized controls for every aspect of RNAi experiments


HiPerFect transfection reagent- can be used in a broad range of cell types

.Human cell lines:� 293� A549� AGS� Caco2� HCT116� HeLa� HeLa S3� HepG2� Huh-7� LNCaP� MCF-7� MDA MB231� U2OS

.Human primary cells:� HUVEC� NHDF

.Mouse cell lines:� NIH/3T3� B16 F1

Human cell lines Mouse cell line

� High efficiency - 100 pM siRNA� Effectiveness – primary cells, suspension cells, macrophages� Low cytotoxicity -minimal impact on cells� Efficient transfection of miRNA mimics or inhibitors.Highly suitable for high throughput screening

http://www.qiagen.com/Products/Applications/Gene-Silencing/#Transfection


Transfection Cell Database- provided by our customers

http://www.qiagen.com/transfection/celllistlist


Assays for real-time RT-PCR- FastLane Cell SYBR Green Kit

Applications:

�Validation of siRNA-mediated gene knockdown

�Screening of potential drugs

�Detection of stimulated gene regulation

� No RNA purification required, significantly saving time

� Just 3 steps from cells to real-time RT-PCR

� High-throughput analysis of 96- and 384-well plates

� Unique gDNA Wipeout Buffer enables detection of RNA only

� Immediate startup using optimized reagents and protocols

From cells to Real-time RT-PCR within minutes

http://www.qiagen.com/Search/FastLane-Cell-SYBR-Green-Kit#orderinginformation


Dicer is required in both the siRNA and miRNApathways

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

Assess RNA Interference Phenotypes- Reporter Assays System

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

Cignal Reporter Assay System

P53 Reporter

+ Dicer siRNA

■ Dual-luciferase & GFP format

■ Plasmid based reporter assay

■ Lentivirial based reporter assayCignal p53 Reporter Assay Kit


Applications – QIAGEN’s siRNA

Application Examples4

1. Medium-throughput siRNA application

-Identify new regulators of apoptosis and chemoresistance

2. Genome-wide siRNA application-Identify human host factors crucial for influenza virus replication


Application 1 – Apoptosis regulation

In total, 650 kinase genes and 222 phosphatase genes were screened

Department of Cell Biology, Harvard Medical School


Application 1 – Experimental workflow

Nat. Cell Biol. 2005. 7, 591: “Sensitized RNAi screen of human kinases and phosphatases identifies

new regulators of apoptosis and chemoresistance”.


Application 1 - Results

Silencing of the 4 survival kinases causes increased apoptosis;2 of 4 these kinases are novel and unknown function (RPS6KL1 and ROR1)

Ref: http://www.ncbi.nlm.nih.gov/pubmed/15864305


Application 2 – Genome-wide screening

Qiagen Human Genome 1.0 and Human Druggable Genome siRNA Set V2.0

Libraries were used to screen

~ 62,000 siRNAs targeting 17,000 annotated genes and 6,000 predicted genes

Molecular Biology Department, Max Planck Institute for Infection Biology, Berlin, Germany


Application 2 : Two-step screen procedure

Step 1:

� A549 human lung epithelial cells were transfected with siRNAs;

� Cells were infected with influenza A H1N1 virus (A/WSN/33).

Step 2:

� Infected cells transferred onto 293T human

embryonic kidney reporter cells;

� Gene knockdown effect assays were performed

Using Immunofluorescence staining and Luciferase

reporter assay


Application 2 – Validation of hits

22,843 human genes Screened

Primary Hits: 287 genes

Validation of Hits: 168 genes

Validation: secondary analysis 11 genes interfered with early events

in virus replication;

7 genes involved in later infection stages

Validation: in vivo analysisCLK1: affect splicing of viral RNAs

P27: affect virus replication

3 parameters:

Luciferase expression;

% infected cells;

Tot. number of infected cells

4 siRNAs /target

Mimics in vivo

Ref: http://www.ncbi.nlm.nih.gov/pubmed/20081832


.https://www.qiagen.com/geneglobe

� Genome-wide human, mouse, and rat libraries

� FlexiPlate & FlexiTube siRNA–100% custom siRNA sets, screening scale

� Most advanced siRNA design, updated database

� Functionally validated siRNA >2000 genes

� AllStar Controls:

Highly characterized negative, positive, and other controls

� Transfection cell database and other resources

� Transfection reagents, qPCR and reporter assays

A Complete solution of RNAi @ QIAGEN


Thank You for Attending!

RNAi Search Portalwww.qiagen.comhttps://www.qiagen.com/geneglobe

Comprehensive RNAi Solution – siRNA

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Si rna 2013

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