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  • 1. 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 . . -1- Sample & Assay Technologies
  • 2. Topics will be Covered Topic I: RNAi High Throughput Screening Applications, Challenges and Solutions; May 6 , 1pm Eastern Time https://www2.gotomeeting.com/register/786104298 Topic II (Today): 1 RNAi Introduction and Challenges 2 Solutions: SureSilencing shRNA Plasmid 3 Key Points to Ensure Successful RNAi 4 Application Examples -2- Sample & Assay Technologies
  • 3. RNA Interference Introduction What are siRNAs? siRNAs are 21–23nt (nucleotide) dsRNA duplexes with symmetric 2–3nt 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 -3- Sample & Assay Technologies
  • 4. RNAi Strategy: siRNA & shRNA siRNA Origin Biogenesis shRNA Synthetic oligonucleotides Introduced into cell by transfection Transient Gene silencing effect Effect on protein Decrease protein levels production Quick transient knockdown, not for long term knocking down; not for enrichment Applications QIAGEN’s Solution FlexiTube siRNA FlexiPlate siRNA 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. SureSilencing shRNA Plasmids The choice of which one to use depends on the question under investigation, the factors such as cell type, time demand, and the need for transient or stable knockdown. -4- Sample & Assay Technologies
  • 5. Why Knockdown the Expression of a Gene? RNAi Gene Function Studies Pathway Interrogation Target Identification and Validation Biomarker and Drug Target Discovery -5- Sample & Assay Technologies
  • 6. 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 -6- Sample & Assay Technologies
  • 7. Solutions @ QIAGEN SureSilencing shRNA Plasmids -7- Sample & Assay Technologies
  • 8. SureSilencing shRNA Plasmids Available for every gene in the Human, Mouse, and Rat genome Guaranteed >70% Knockdown by at least 2 different shRNAs 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 Customer’s success story: http://www.sabiosciences.com/RNAipublication.php -8- Sample & Assay Technologies
  • 9. SureSilencing shRNA – How it works 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. -9- Sample & Assay Technologies
  • 10. SureSilencing shRNA Plasmids – Mechanism U1 promoter transcribes a moderate amount of shRNA The ampicillin resistance marker and bacterial origin of replication permit amplification for a lifetime supply of plasmid FACS enrichment GFP Antibiotic-resistance markers: Stable cell line development Hyg Neo Puro Enrich or Select: 4 Markers: GFP, Nyomycin, Hygromycin, and Puromycin Multiple Designs: 4 Designs for each gene – each sequence targets different region - 10 - Sample & Assay Technologies
  • 11. The Best shRNA Design Algorithm Download White Paper “Did Your RNAi Experiment Work?!” http://www.sabiosciences.com/validaternai.pdf - 11 - Sample & Assay Technologies
  • 12. 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. - 12 - Sample & Assay Technologies
  • 13. 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 Successful Designs Success Rate (%) Genes Tested Successful Genes Success Rate (%) SABio’s Set 329 221 67.2 86 74 86.0 The RNAi Consortium 2561, 5422 971, 172 381, 312 53 40 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. - 13 - Sample & Assay Technologies
  • 14. 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 - 14 - Sample & Assay Technologies
  • 15. 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 - 15 - Sample & Assay Technologies
  • 16. SureSilencing shRNA plasmids – search portal Search by pathway or diseases - 16 - Sample & Assay Technologies
  • 17. 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 - 17 - Sample & Assay Technologies
  • 18. 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 48hr Incubation - 18 - Sample & Assay Technologies
  • 19. Workflow for shRNA plasmids knockdown Assay gene knockdown effect 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 - 19 - Sample & Assay Technologies
  • 20. 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. - 20 - Sample & Assay Technologies
  • 21. shRNA experiment optimization Key points to ensure successful RNAi Controls Enrichment Selection Validation - 21 - Sample & Assay Technologies
  • 22. Optimization #1 - controls Untreated cells: Use normal cells in a normal culture condition as a pure background 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 - 22 - Sample & Assay Technologies
  • 23. 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 Percent Knockdown Pre-Sorted Population (%) Knockdown Sorted Population(%) Knockdown 37 71.8 (69.7, 73.8) 52 70.8 (68.4, 73.0) PRKCA Protein Kinase C alpha TP53 Tumor protein p53 - 23 - Sample & Assay Technologies
  • 24. Optimization #3 - selection Selection using entire pool 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 Potential challenge when using pooled population Initial stably antibiotic selected whole pool population appears to fail, due to: Individual Stably Selected Clones -Random integration sites affect shRNA expression and knockdown efficiency -Average knockdown efficiency of all integration sites is seen, some better than others 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 - 24 - Sample & Assay Technologies
  • 25. Optimization #4 - validation To ensure >70% knockdown efficiency: Transfection Efficiency (TE): >=80% Biological Sample Consistency: 3 Validation – qPCR at the mRNA level • • • PCR Reproducibility PCR Amplification Efficiency Site-specific primer may be necessary for some genes 100 Knocking down efficiency (% ) • • 90 80 70 60 default primer 50 site specific primer 40 30 20 10 0 A B C D E Designs 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. - 25 - Sample & Assay Technologies
  • 26. shRNA Case Studies Application Examples – Case Studies - 26 - Sample & Assay Technologies
  • 27. shRNA Case Study 1 Validation of STAT3 RNAi Design in-house Target GOI (Gene of interest): STAT3 (Signal transducer and activator of transcription 3) Assay method: Real-time RT-PCR Cell model: 293H cells Control HKG (Housekeeping gene): ACTB (Beta actin) - 27 - Sample & Assay Technologies
  • 28. shRNA Case Study #1– Experimental Workflow Validation of STAT3 RNAi Design Transfection Grade shRNAs Cell Culture: 293H cells were cultured in D-MEM with 10% FBS and 1x non-essential amino acids for no more than 15 passages. 3 x 4 GOI + 3 x 1NC = 15 shRN A1 shRN A2 shRN A3 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. shRN A4 Check transfection efficiency by GFP expression using fluorescence microscopy Isolate total RNA: Isolated total RNA after 48 hrs Isolate RNA - 28 - Sample & Assay Technologies
  • 29. 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 - 29 - Analyze data & report Sample & Assay Technologies
  • 30. 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 STAT3-1 STAT3-2 Real-Time PCR Result for STAT3 shRNA STAT3-3 STAT3-4 2 24.11 24.30 24.28 3 24.08 24.29 24.19 1 24.44 24.56 24.57 2 24.18 24.31 24.32 3 24.18 24.32 24.28 1 31.54 31.58 31.92 2 22.67 22.78 22.82 3 23.09 23.33 23.28 1 24.31 24.46 24.47 2 24.51 24.55 24.64 3 24.26 24.35 24.29 1 28.96 28.83 28.87 2 23.03 22.94 23.00 3 22.90 22.82 22.83 24.04 24.23 24.19 24.52 24.27 24.26 31.68 22.76 23.23 24.41 24.57 24.30 28.89 22.99 22.85 0.05 0.10 0.11 0.07 0.08 0.07 0.21 0.08 0.13 0.09 0.07 0.05 0.07 0.05 0.04 OK 17.65 17.83 17.72 OK 17.74 17.96 17.81 OK 17.62 17.70 17.66 OK 17.79 17.86 17.77 OK 17.82 17.87 17.75 OK 17.81 17.74 17.83 OK 26.38 26.39 26.45 OK 18.03 17.93 18.00 OK 18.46 18.50 18.32 OK 17.85 17.86 17.69 OK 17.87 17.94 17.84 OK 17.68 17.81 17.63 OK 25.05 24.90 24.97 OK 19.18 19.10 19.15 OK 19.15 19.12 19.16 17.73 17.84 17.66 17.81 17.81 17.79 26.41 17.99 18.43 17.80 17.88 17.71 24.97 19.14 19.14 GOI- STAT3 HKG - ACTB 0.09 OK 0.11 OK 0.04 OK 0.05 OK 0.06 OK 0.05 0.04 OK OK 0.09 OK 0.10 OK 0.05 OK 0.09 OK 0.08 OK 0.04 OK 0.02 OK 6.39 6.53 6.72 6.46 6.47 5.27 4.77 4.81 6.61 6.68 6.59 3.91 3.85 3.71 0.10 0.15 0.11 0.09 0.10 0.09 0.21 0.09 0.16 0.13 0.08 0.10 0.10 0.06 0.05 6.41 6.55 SD ∆Ct BIO 0.11 Overall Mean SD ∆Ct 0.13 ∆∆Ct STAT3-1 STAT3-2 STAT3-3 STAT3-4 0.05 OK 6.30 Average ∆Ct Overall SD ∆∆Ct QC 3 Percent of Control Percent Knock Down + SD - SD Report: Negative Control 1 24.00 24.09 24.02 4.95 6.63 3.82 0.15 0.28 0.05 0.11 0.16 0.30 0.08 0.11 2.59 2.72 1.13 2.81 0.18 0.19 0.32 0.14 OK 0.17 83.34 1.91 2.15 OK 0.15 84.87 1.90 2.17 OK 0.46 54.24 9.02 11.24 OK 0.14 85.72 1.30 1.43 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 ) ) ) ) - 30 - Design Successful Successful Mediocre Successful Sample & Assay Technologies
  • 31. shRNA Case Study #2 Case Study 2 – Published by customers - 31 - Sample & Assay Technologies
  • 32. shRNA Case Study #2 – Cancer Research Study: The role of PELP1/MNAR signaling in Ovarian Tumorigenesis PELP1/MNAR (Proline-, glutamic acid–, and leucine–rich 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 - 32 - Sample & Assay Technologies
  • 33. 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. - 33 - Sample & Assay Technologies
  • 34. 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 - 34 - Sample & Assay Technologies
  • 35. SureSilencing shRNAs for Every Gene - A complete system for RNAi from QIAGEN shRNAs Analysis Purification Transfection 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 - 35 - Sample & Assay Technologies
  • 36. Assessing RNA Interference Phenotypes - 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 What’s 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. - 36 - Sample & Assay Technologies
  • 37. 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 Thank You for Attending! Search Portal http://sabioscience.com/RNAi.php www.qiagen.com www.GeneGlobe.com Call 888-503-3187 to order Email: support@SABioscience.com Wei Cao: wei.cao@qiagen.com - 37 - Sample & Assay Technologies