A Comprehensive Solution for RNAi

Transcription

1 A Comprehensive Solution for RNAi RNAi Screen Application, Challenges and Solutions Wei Cao, Ph.D

2 Topics will be covered 1 Overview of RNAi 2 RNAi application and challenges 3 sirna screen 4 Case studies - 2 -

3 RNA interference introduction 1 Overview of RNAi Brief history of RNAi Mechanism of RNAi - 3 -

4 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

5 What is RNAi? RNAi Properties Natural biological mechanism Resistance to viral infection Regulation of gene expression Maintenance of heterochromatin Post-transcriptional gene regulation Initiated by double-stranded RNA (dsrna), processed by DICER Effector is ~ 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

6 The sirna and mirna pathways sirna Pathway sirna mirna Pathway!? From Meister & Tuschl,

7 sirnas and mirnas: How are they different?.sirna: mirna:. Target mrna for degradation mrna degraded Effect: Reduced level of mrna Reduced level of protein Perfect complementarity to target Not endogenous to mammalian cells Precursor: none in mammalian cells Inhibits translation of protein mrna intact Effect: Normal level of mrna Reduced level of protein Typically multiple mismatches to target Endogenous to mammalian cells Precursor: ~70mer ssrna - 7 -

8 RNAi as a tool for functional analysis 2 RNAi Application Challenges of RNAi screen Key issues for sirna screening Establish an effective HT sirna experiment - 8 -

9 RNAi as a tool for functional analysis RNAi Decoding gene functions Cellular signaling pathway interrogation Target identification Target validation Drug discovery and disease therapy (Infectious diseases and cancer) Mohr S, Bakal C, Perrimon N. Genomic screening with RNAi: results and challenges. Annu Rev Biochem. 2010;79:

10 RNAi Screen Application - sirna sirna is the reagent choice for large scale functional analysis Ready to use, QC d and stable molecules Effective design rules allow potent silencing Efficient transfection into many cell types High throughput (1000 s/day) Ability to label and track sirna Modifications easily incorporated (stability, enhanced transfer, potency, etc.)

11 RNAi screen challenges Intrinsic limits on the specificity of RNAi Off-Target effect (OTE) is #1 Challenge, can come from: 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 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

12 Critical factors for successful sirna screen Maximize on-target effects, minimize off-targets effects Effective and specific sirna design sirna Delivery - Optimize transfection condition Validation is always a key Speficity must be confirmed with multiple sirnas Controls Appropriate negative and positive control experiments

13 sirna specificity and potency - design 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!

14 Parameters to optimize transfection Every cell line is different Parameters that need to be optimized for each cell line: Cell culture density Passage number Amount of transfection reagent Amount of sirnas Complex incubation time on cells Optimal time point of analysis Proper controls

15 Optimization of transfection condition Titration of sirna and reagents Optimize for the best results: Vary amount of reagent and sirnas Vary reagent-to-sirna ratio Complex incubation time and analysis, adjust: Incubation time of cells with transfection complexes Optimal time point of analysis Target gene, analysis method

16 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 non-specific sequence, providing a useful reference for interpretation of knockdown. Positive sirna control: Ubiquitously expressed (e.g. lamin, actin, g3pdh) Assay-specific positive control: Confirm assay is working (when screening for phenotype) Use of negative controls - Scrambled or Non-targeting Negative controls The best available tool for sequence-independent off-target effects Several negative controls should be tested with each new assay or cell combination To reflect cellular, undisturbed baseline Whether certain assays are more prone to be affected by off-target effects Monitor specific and endogenous-based cell readouts or multiple parallel analysis

17 Validation of hits - large scale sirna screen 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 of phenotype to target gene Redundancy: Confirms specificity of phenotype by multiple independent sirnas Rescue by cdna lacking targeting sequence (eg 5 UTR) Key to success: Validation

18 Validation: Specificity must be confirmed with multiple sirnas % Normalized Survival VEGF B duplex 1 HeLa S3 (1) HeLa S3 (2) HEK 293 Hep G2 MCF-7 VEGF B duplex 2 HeLa S3 (1) HeLa S3 (2) HEK 293 Hep G2 MCF-7 VEGF B duplex 3 HeLa S3 (1) HeLa S3 (2) HEK 293 Hep G2 MCF

19 Minimize OTE: Commentary in Nature Methods: The Two Rs Solution 1: Redundancy Solution 2: Rescue 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):

20 Minimize OTE: Commentary in Nature Methods: The Two Rs Approach Redundancy Rescue Definition Multiple, distinct silencing of the same gene generates the same phenotype The RNAi-induced phenotype is countered by expression of a functional version of the target gene that is resistant to the silencing reagent.probability very low that several sirnas/shrnas with completely distinct sequences will share sequence-dependent OTE.Technically challenging Comments.Cannot be done on a large scale Thus, confirmation with redundancy offers the most straightforward and compelling way of demonstrating specificity in large-scale screens 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):

21 Minimize OTE: Commentary in Nature Methods How much redundancy? How many replicates depends on the following: Design of sirna Organism, or model used Biologic pathway Analysis method.at least two distinct sirna sequences are recommended for a result to be valid Besides replicates, there are other methods to increase your confidence: Monitor multiple markers Examine kinetics via multiple time points Compare phenotypes from genes in a pathway At the least, do these in secondary or tertiary screens Nybakken K, Vokes SA, Lin TY, McMahon AP, Perrimon N. A genome-wide RNA interference screen in Drosophila melanogaster cells for new components of the Hh signaling pathway. Nat Genet (12):

22 3 sirna Screen Solutions QIAGEN provides would-class RNAi solutions: Evolution of sirna design QIAGEN s sirna validation project Screening solutions: Flexiplate sirna HiPerfect transfection reagent

23 Evolution of sirna design Phase 1 the Tuschl Rules in 2002, 50% active, AA(N)19, moderate G.C, a simple BLAST 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; Hall et al. Nature biotechnology, July 2005, Phase 4 HP OnGuard sirna 2006/ 2008 Latest developments: addressing mirna related off-target effects Maximal efficiency + minimal off-target effects Lim et al. 2005, Lewis et al. 2005, Saxena et al

24 Evolution of sirna design 2002: The Tuschl 120 Rules AA(N)19, moderate G/C, a simple BLAST:50% active ; 2004: Tm rulesasymmetry; 75% active ; % remaining mrna % 75% 2005: BioPred Si, neural-network algorithm trained to select potent sirna; ~83% active duplexes per target, 25 targets 50% 1. Schwarz, et. al. Cell, Vol. 115, Hall. et al. Nature Biotechnology July

25 Reducing mirna related off-target effects - Seed Region Analysis 3 UTR-Seed Region Analysis CDS sirnas that bind like mirna 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

26 Asymmetry Design: a primary feature of successful sirna design, unequal stability of base pairs at the 5 ends to ensure high efficiency and reduce off-target effect; Neural-network Technology: BioPred SI neural-network algorithm, an algorithm trained to select potent sirna based on large RNAi data set; SNP Avoidance: optimized to avoid known single nucleotide polymorphisms (SNPs) using RefSNP database; Affymetrix GeneChip Analysis Homology Analysis 3 UTR-seed Region Analysis: design sirnas with minimal complementation to the 3 - UTR of all the genes to improve the specificity of sirna; HP OnGuard sirna design algorithm - Highest knockdown efficiency and specificity Interferon Motif Avoidance*: screened for multiple sequence motifs known to result in an interferon response sirnas ranked by efficacy, specificity, splice variant coverage, etc, by a QIAGEN algorithm, The top 4 sirnas are included in the QIAGEN collection Download more information of HP OnGuard sirna Design at: *Hornung et al., Nature Medicine, 2005; Judge et al., Nature Biotech,

27 QIAGEN sirna validation project -The world largest sirna validation project QIAGEN algorithm based on ~ 8000 sirnas (3000 genes) > Largest validated sirna set: 3700 sirnas Algorithm feeds validation, validation feeds algorithm.allow researchers to focus on the results of RNAi, not the QC of sirna Functional evaluation performed by QIAGEN Reduction of unknowns.no need to purchase non-functional or poorly functional sirna.for many genes, QIAGEN has 2 or more validated sirnas per target - essential independent confirmation of phenotype. provides the Krüger et al., 2007; Insights into Effective RNAi Gained from Large-Scale sirna Validation Screening. Oligonucleotides 17:

28 RNAi Product Portfolio Low Throughput RNAi Medium to High Throughput RNAi Predesigned FlexiTube sirna FlexiTube GeneSolution FlexiTube sirna Premix FlexiPlate sirna AllStars Control sirna Custom synthesis HP Custom sirna Vector based SureSilencing shplasmids Register shrna webinar: Jan

29 FlexiblePlate high throughput - Customizable and economical screening solution FlexiPlate sirna Custom sirna sets for customerspecified genes and sirna controls 96 or 384-well format for HT screening applications Maximum flexibility to 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

30 FlexiTube sirna low throughput: 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

31 AllStars RNAi controls AllStars Transfection Controls AllStars Negative Controls AllStars Positive Controls AllStars Downstream Controls Transfection reagents for assessment of transfection efficiency Highly validated nonsilencing sirna Routine positive control for optimal conditions, MAPK1, cell death RT qpcr primer sets for knockdown analysis AllStars Interferon Controls AllStars Reporter Controls RT qpcr primer sets for detection of interferon response sirnas for knockdown of reporter genes Key pathway gene controls sirnas targeting genes central to important pathways

32 HiPerFect transfection reagent.efficient transfection with low sirna concentrations pm sirna Minimizing the risk of off-target effects.fastforward Transfection same day transfection Suitable for high-throughput RNAi and reverse transfection Easy and simple protocol (also for 384-well format).effective transfection of primary and suspension cells.minimal cytotoxicity/ minimal impact on cells Reduces risk of nonspecific side effects High cell viability.highly suitable for high throughput/screening purposes Suitable for automation procedures Stable and reproducible.new! HiPerFect HTS reagent

33 HiPerFect transfection reagent - can be used in a broad range of cell types Human cell lines Mouse cell line.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 F1-33 -

34 TransFect Protocol Database The bench-ready protocol Protocols for DNA & sirna transfection, cell and plate format specific

35 Case studies QIAGEN s sirna application 4 Case Studies 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

36 Case Study 1 Regulation of apoptosis Department of Cell Biology, Harvard Medical School In total, 650 kinase genes and 222 phosphatase genes were screened

37 Case Study 1 Experimental workflow

38 Case Study 1 - Results Silencing of the 4 survival kinases causes increased apoptosis; 2 of 4 these kinases are novel and unknown function (RPS6KL1 and ROR1) Nat. Cell Biol , 591: Sensitized RNAi screen of human kinases and phosphatases identifies new regulators of apoptosis and chemoresistance

39 Case Study 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

40 Case Study 2 : Two-step screen procedure Step 1: A549 human lung epithelial cells were transfected with sirnas 48 h; Cells were infected with influenza A H1N1 virus (A/WSN/33), then stained with a virus specific antibody at 24 h after infection to monitor cell infection rates. Step 2: Virus supernatants transferred onto 293T human embryonic kidney reporter cells, containing an inducible influenza-virus-specific luciferase (FlaA); Assay reliability confirmed with an sirna-directed against influenza virus nucleoprotein (NP), assessed by immunofluorescence staining and the luciferase reporter assay

41 Case Study 2 Validation of hits 22,843 human genes Screened Primary Hits: 287 genes Identification of primary hits: 3 parameters a. Luciferase expression b. The percentage of infected cells c. The total number of infected cells. Validation of Hits: 168 genes 119 & 121, 72 common Validation of hits: Replication of 2 strains: Influenza A A/WSN/33 (HIN1) Swine-origin influenza A/Hamburg/04/2009 (H1N1) 4 different sirnas were used Life-stage relevance 18 genes Extended to Avian-origin influenza A virus of H5N1 11 genes interfered with early events in virus replication; 7 genes involved in later infection stages In vivo validation confirmed: CLK1: affect splicing of viral RNAs P27: affect virus replication

42 A Complete solution of QIAGEN Validation of knockdown by qpcr Wide dynamic range in SYBR Green based real-time RT-PCR

43 A Complete solution of QIAGEN Reporter Assay System Easy to transfect cell lines Cignal Reporter Assays Two systems: dual-luciferase and GFP reporter systems; High performance: sensitive, reproducible, specific and signal to noise ratio; Primary cells, stem cells, and difficult to transfect cell lines Cignal Lenti Reporter Assays Ready to transduce Transduce any cell type Minimal cellular stress Wide Application: suitable for transient experiments and stable pathway sensor cells Versatility: perform endpoint format assays or dynamic live cell assays; Convenience: transfection ready, with positive and negative controls

44 Steps to ensure successful RNAi screen Key steps involved in RNAi screen: sirna or shrna design: Optimal RNAi design is essential Delivery: Optimal transfection and culture conditions Controls: Must have appropriate positive and negative control experiments Validation of knockdown: Must have a reliable method to evaluate the effects caused by RNAi-mediated gene knockdown; and be validated by at least 2 RNAis

45 Comprehensive RNAi Solution sirna FlexiPlate and Flexitube sirna Search Portal SureSilencing shrna Plasmid Search Portal SABiosciences.com