Sample & Assay Technologies -1- A Comprehensive Solution for RNAi HT RNAi Screening Application, Challenges and Solutions Any Questions ??? Ask now or contact Technical Support [email protected]1-888-503-3187 International customers: [email protected]Webinar related questions: [email protected]
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Sample & Assay Technologies- 1 -
A Comprehensive Solution for RNAi
HT RNAi Screening
Application, Challenges and Solutions
Any Questions ???Ask now or contact Technical Support
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?
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The Biological question or hypothesis
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• 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
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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)
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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
siRNA is the choice for large scale functional analy sis
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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
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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
� Assay-specific positive control : Confirm assay is working (when screening for phenotype)
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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
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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
HeLa S3 (1)HeLa S3 (2)HEK 293Hep G2MCF-7
HeLa S3 (1)HeLa S3 (2)HEK 293Hep G2MCF-7
% Normalized Survival
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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
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� 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.
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QIAGEN provides world-class RNAi solutions:
� QIAGEN’s siRNA design and validation
� Screening solutions: Flexiplate siRNA
� HiPerfect transfection reagent
3 siRNA Solutions @ QIAGEN
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� 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
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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’
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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
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A Complete solution of RNAi @ QIAGEN
Integration of every step of gene silencing workflow
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
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HiPerFect transfection reagent- can be used in a broad range of cell types