Si rna 2013


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Si rna 2013

  1. 1. A Comprehensive Solution for RNAi HT RNAi Screening Application, Challenges and Solutions Any Questions ??? Ask now or contact Technical Support 1-888-503-3187 International customers: Webinar related questions: Qiawebinars@QIAGEN.COM -1- Sample & Assay Technologies
  2. 2. Topics will be covered 1 Overview of RNAi 2 RNAi application and challenges 3 siRNA solutions @QIAGEN 4 Application examples -2- Sample & Assay Technologies
  3. 3. RNA interference: a natural phenomenon Discovery 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. -3- Sample & Assay Technologies
  4. 4. What is RNA interference? - The RNAi Pathways RNAi: a research tool RNAi 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 -4- Sample & Assay Technologies
  5. 5. The siRNA and miRNA pathways are related miRNA Pathway siRNA Pathway siRNA !? From Meister & Tuschl, 2005 -5- Sample & Assay Technologies
  6. 6. RNAi as a tool for functional analysis 1 Overview of RNAi RNAi Application 2 RNAi applications and challenges 3 siRNA solutions @QIAGEN 4 Application examples -6- Sample & Assay Technologies
  7. 7. RNAi as a tool for functional analysis “Learning by Breaking” - simply removing a gene and looking at the effect Decode gene functions - functional genomics Interrogate cellular signaling pathway-system biology Target identification and validation Drug discovery and disease therapy (Infectious diseases and cancer) What types of questions can be answered by RNAi screens ? Mohr S, Bakal C, Perrimon N. “Genomic screening with RNAi: results and challenges. “Annu Rev Biochem. 2010;79:37-64 -7- Sample & Assay Technologies
  8. 8. The Biological question or hypothesis • 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 kinaseSTAT transcription factors • … • Identify an unknown protein Decide which method to use: genome-wide screens or more focused screens -8- Sample & Assay Technologies
  9. 9. RNAi HTS platform 3 steps Transfection Transfer to 384 or 96 well plates dsRNA library stored in 96-well plates Ready-toscreen 384well plate Add cells transfection 3-7 days incubation to ensure protein depletion Transcriptional-Luciferase reporter assays Protein modification (e.g. phospho-specific antibodies) -9- Automated Microscopy (GFP, fluorescent dyes or antibody labeling) Sample & Assay Technologies
  10. 10. 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 - 10 - Sample & Assay Technologies
  11. 11. 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 - 11 - Sample & Assay Technologies
  12. 12. 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! - 12 - Sample & Assay Technologies
  13. 13. Choice of siRNA libraries siRNA is the choice for large scale functional analysis 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.) - 13 - Sample & Assay Technologies
  14. 14. Optimization of transfection condition Every cell line is different Parameters 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 - 14 - Sample & Assay Technologies
  15. 15. 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) - 15 - Sample & Assay Technologies
  16. 16. 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) There should be a clear correlation between protein depletion and phenotypic severity Key to success: Validation - 16 - Sample & Assay Technologies
  17. 17. Validation: Specificity must be confirmed with multiple siRNAs & multiple cell lines % Normalized Survival 0 VEGF B duplex 1 60 80 100 HeLa S3 (1) HeLa S3 (2) HEK 293 Hep G2 MCF-7 VEGF B duplex 3 40 HeLa S3 (1) HeLa S3 (2) HEK 293 Hep G2 MCF-7 VEGF B duplex 2 20 HeLa S3 (1) HeLa S3 (2) HEK 293 Hep G2 MCF-7 - 17 - Sample & Assay Technologies
  18. 18. Validation: Redundancy and Rescue - Commentary in Nature Methods: “The Two Rs” Solution 2: Rescue Solution 1: Redundancy Reintroduce an RNAi-resistant version of the target gene product into depleted cells and show recovery of function . “… the only ways of adequately addressing sequence-dependent offtarget effects within RNAi experiments themselves are the ‘the two Rs’.” Specificity Must be Confirmed with Multiple siRNAs ! Echeverri et al. (2006), Minimizing the risk of reporting false positives in large-scale RNAi screens. Nat Methods, 3(10):777-9 - 18 - Sample & Assay Technologies
  19. 19. Validation: Secondary analysis 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 Sonia S. and Anjana R. “RNAi screening: tips and techniques”Nat Immunol 2009 10(8):799-804. - 19 - Sample & Assay Technologies
  20. 20. 3 siRNA Solutions @ QIAGEN QIAGEN provides world-class RNAi solutions: QIAGEN’s siRNA design and validation Screening solutions: Flexiplate siRNA HiPerfect transfection reagent - 20 - Sample & Assay Technologies
  21. 21. Evolution of siRNA design @ QIAGEN 120 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 % remaining mRNA 100 80 60 83% 75% 40 20 2004/2005; 83% active 50% Phase 4 – HP OnGuard siRNA 2006/ 2008 0 11 Latest developments: addressing miRNA related off-Target effects 11 21 31 41 51 61 10 20 30 40 50 60 7071 8081 9091 100 4 duplexes per target, 25 targets Maximal efficiency + minimal offtarget effects Lim et al. 2005, Lewis et al. 2005, Saxena et al. 2003 Schwarz , et. al. Cell, Vol. 115, 2003 Hall. et al. Nature Biotechnology July 2005 - 21 - Sample & Assay Technologies
  22. 22. Reduce miRNA related off-target effects - Seed Region Analysis 3’UTR-Seed Region Analysis siRNAs that bind like miRNA CDS 5’ AAAAA 3’ 3`UTR 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 - 22 - Sample & Assay Technologies
  23. 23. HP OnGuard siRNA design algorithm - Highest knockdown efficiency and specificity Neural-network technology Up-to-date siRNA target sequences The world’s largest siRNA validation project: >3,700 siRNA Asymmetry Homology analysis HP OnGuard siRNA Design SNP avoidance Interferon motif Affymetrix GeneChip analysis >2 siRNA/target 3' UTR/seed avoidance region analysis For more information of HP OnGuard siRNA Design: - 23 - Sample & Assay Technologies
  24. 24. A Complete solution of RNAi @ QIAGEN Integration of every step of gene silencing workflow Step 1 Step 2 Step 3 Step 4 - 24 - Sample & Assay Technologies
  25. 25. RNAi Product Portfolio Predesigned Low Throughput RNAi FlexiTube siRNA Medium to High Throughput RNAi FlexiPlate siRNA FlexiTube GeneSolution FlexiTube siRNA Premix Vector based Custom synthesis AllStars Control siRNA HP Custom siRNA SureSilencing shPlasmids shRNA webinar: April 19, 1-2pm EDT - 25 - Sample & Assay Technologies
  26. 26. High throughput screening: FlexiblePlate - Customizable and economical screening solution FlexiPlate siRNA Custom siRNA sets for customerspecified 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 - 26 - Sample & Assay Technologies
  27. 27. 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 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 - 27 - Sample & Assay Technologies
  28. 28. AllStars RNAi controls Extensively characterized controls for every aspect of RNAi experiments 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 - 28 - Sample & Assay Technologies
  29. 29. HiPerFect transfection reagent - can be used in a broad range of cell types 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 . Human cell lines 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 . Mouse cell line . . - 29 - Sample & Assay Technologies
  30. 30. Transfection Cell Database - provided by our customers - 30 - Sample & Assay Technologies
  31. 31. Assays for real-time RT-PCR - FastLane Cell SYBR Green Kit From cells to Real-time RT-PCR within minutes 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 - 31 - Sample & Assay Technologies
  32. 32. Assess RNA Interference Phenotypes - Reporter Assays System Cignal Reporter Assay System ■ Dual-luciferase & GFP format ■ Plasmid based reporter assay Cignal p53 Reporter Assay Kit ■ Lentivirial based reporter assay Dicer is required in both the siRNA and miRNA pathways What’s the phenotypic effect of Dicer knock down on p53 signaling? P53 Reporter + Dicer siRNA Conclusion: The regulation of p53 is tightly controlled by miRNA and/or siRNA processing. - 32 - Sample & Assay Technologies
  33. 33. Applications – QIAGEN’s siRNA 4 Application Examples 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 - 33 - Sample & Assay Technologies
  34. 34. Application 1 – Apoptosis regulation Department of Cell Biology, Harvard Medical School In total, 650 kinase genes and 222 phosphatase genes were screened - 34 - Sample & Assay Technologies
  35. 35. 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”. - 35 - Sample & Assay Technologies
  36. 36. 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: - 36 - Sample & Assay Technologies
  37. 37. Application 2 – Genome-wide screening Molecular Biology Department, Max Planck Institute for Infection Biology, Berlin, Germany 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 - 37 - Sample & Assay Technologies
  38. 38. 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 - 38 - Sample & Assay Technologies
  39. 39. Application 2 – Validation of hits 3 parameters: 22,843 human genes Screened Luciferase expression; % infected cells; Primary Hits: 287 genes Tot. number of infected cells Validation of Hits: 168 genes 4 siRNAs /target Validation: secondary analysis 11 genes interfered with early events in virus replication; 7 genes involved in later infection stages Validation: in vivo analysis Mimics in vivo CLK1: affect splicing of viral RNAs P27: affect virus replication Ref: - 39 - Sample & Assay Technologies
  40. 40. A Complete solution of RNAi @ QIAGEN . 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 - 40 - Sample & Assay Technologies
  41. 41. Comprehensive RNAi Solution – siRNA Thank You for Attending! RNAi Search Portal - 41 - Sample & Assay Technologies