Tpa 2013

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Tpa 2013

  1. 1. SureFIND™ Transcriptome PCR Arrays cDNA Panels to Accelerate Discovery Discover which miRNA, transcription factor or signaling pathway is regulating your favorite gene’s expression. Samuel J. Rulli, Ph. D. Samuel.Rulli@QIAGEN.com Sample & Assay Technologies
  2. 2. Webinar Topics . Transcriptional Regulation and Mechanisms What regulates your gene’s expression? Techniques . What is a Transcriptome PCR Array? General Description Protocol Overview . Discovery in an Afternoon Application Example . Data Analysis and Technical Tips Easy to use Downloadable Excel Templates . What’s Next and Summary Follow-up Experiments -2- Sample & Assay Technologies
  3. 3. What regulates your gene? miRNA Upstream Pathway Regulators mRNA GOI DNA Transcriptional Factors -3- Sample & Assay Technologies
  4. 4. How do you identify those regulators? Bioinformatics Overexpression . . Promoter Analysis Chemical Treatments . . × mRNA RNAi knockdown Molecular Techniques . . × DNA -4- Sample & Assay Technologies
  5. 5. So who knows what regulates your gene-of-interest? -5- Sample & Assay Technologies
  6. 6. What regulates your gene? miRNA Upstream Pathway Regulators mRNA DNA Transcriptional Factors -6- Sample & Assay Technologies
  7. 7. Approach to Identifying Regulators miRNA 1 miRNA mimic 2 3 4 1) Treat Cells 2) Isolate RNA 3) First Strand cDNA Synthesis 4) qPCR for Gene Expression -7- Sample & Assay Technologies
  8. 8. I can do this once or twice… Reagent miRNA mimic Price $150 - $240 Price for 10 / 50 / 90 $1500 - $2400 / $7500 - $12000 / $13500- $21600 -8- Sample & Assay Technologies
  9. 9. Webinar Topics . Transcriptional Regulation and Mechanisms What regulates your gene’s expression? Techniques . What is a Transcriptome PCR Array? General Description Protocol Overview . Discovery in an Afternoon Application Example . Data Analysis and Technical Tips Easy to use Downloadable Excel Templates . What’s Next and Summary Follow-up Experiments -9- Sample & Assay Technologies
  10. 10. What is a Transcriptome PCR Array? Transcriptome PCR Arrays are ready-to-use cDNA panels for discovering the miRNAs, Pathways or Transcription Factors that regulate the expression of any gene. . Each Transcriptome PCR Array is a panel of Transcriptomes or cDNAs: 90 Experimental cDNA samples 4 Vehicle Control cDNA samples 2 PCR Controls (only for SYBR green reactions) . Each experimental cDNA sample is derived from cells treated with a different treatment. . - 10 - Sample & Assay Technologies
  11. 11. Example Plate cDNA Sample 1 cDNA Sample 2 cDNA Sample 3 cDNA Sample 4 cDNA Sample 5 cDNA Sample 6 - 11 - Sample & Assay Technologies
  12. 12. What is needed for the Protocol? What’s in the box? One Transcriptome PCR Array miRNA mimics TF knockdown, Chemical Inhibitors of key Pathway Proteins . . The Researcher Must Supply: . PCR Thermocycler . Micropipettor qPCR Master Mix qPCR Assay (either SYBR® green or Probe-based Assays) Computer with Microsoft Excel for Data Analysis - 12 - Sample & Assay Technologies
  13. 13. Protocol - 13 - Sample & Assay Technologies
  14. 14. Webinar Topics . Transcriptional Regulation and Mechanisms What regulates your gene’s expression? Techniques . What is a Transcriptome PCR Array? General Description Protocol Overview . Discovery in an Afternoon Application Example . Data Analysis and Technical Tips Easy to use Downloadable Excel Templates . What’s Next and Summary Follow-up Experiments - 14 - Sample & Assay Technologies
  15. 15. Application Example: miRNA regulation of INPPL1 INPPL1, also known as SHIP2, is a SH2-containing 5'-inositol phosphatase . Converts PIP3 to PtdIns(3,4)P2, which negatively regulates PI3K signaling . Linked to Carcinogenesis . - 15 - Sample & Assay Technologies
  16. 16. Canonical pathway of microRNA (miRNA) biogenesis Transcribed by RNA Polymerase II as a long primary transcript (pri-miRNAs), which may contain more than one miRNA. In the nucleus, pri-miRNAs are processed to hairpin-like pre-miRNAs by RNAse III-like enzyme Drosha. Pre-miRNAs are then exported to the cytosol by Exportin 5. In the cytosol RNAse III-like Dicer processes these precursors to mature miRNAs. These miRNAs are incorporated in RISC. miRNAs with high homology to the target mRNA lead to mRNA cleavage. miRNAs with imperfect base pairing to the target mRNA lead to translational repression and/or mRNA degradation. - 16 - Sample & Assay Technologies
  17. 17. How to determine miRNA-mRNA interactions Algorithms for predicting miRNA-mRNA interaction Target Prediction is based on: Bioinformatics . Pitfalls of using prediction algorithms: Large number of candidate mRNAs for a given miRNA May not incorporate all miRNA targeting possibilities Different algorithms produce different target lists Potential for false positive rate of prediction . Seed region match Position in 3’ UTR Cross species conservation Central sequence homology Wet lab research Empirical evidence from microarrays Reporter systems Prediction Algorithm Website TargetScan http://www.targetscan.org/ Pictar http://pictar.mdc-berlin.de/ MicroCosm Targets http://www.ebi.ac.uk/enright-srv/microcosm/htdocs/targets/v5/ DIANA http://diana.cslab.ece.ntua.gr/microT/ miRANDA http://www.microrna.org/microrna/home.do TarBase (experimentally supported) http://diana.cslab.ece.ntua.gr/tarbase/ - 17 - Sample & Assay Technologies
  18. 18. How to determine miRNA-mRNA interactions Experimental techniques miRNA Target Screening . Gene expression analysis (inferred targets) RNA-seq Microarrays qPCR Immunoprecipitation (direct targets) HITS-CLIP PAR-CLIP Biotin tagged miRNA Gene-Specific Validation . qPCR Luciferase reporter assays Western blot 5’ rapid identification of cDNA ends (5’ RLM-RACE) Other techniques . Parallel analysis of RNA ends (modified 5’ RLM-RACE) Reverse transcription of targets Image From Chi, S.W., et al, Nature 2009, 13, 479-486 - 18 - Sample & Assay Technologies
  19. 19. Site Predictions for INPPL1 TargetScan PicTar miRANDA miR-10 miR-184 miR-18a miR-135 miR-205 miR-18b miR-146 miR-342-3p miR-184 miR-642 miR-18ab miR-671-3p miR-205 miR-27ab miR-29abc - 19 - Sample & Assay Technologies
  20. 20. Manipulating miRNA function - 20 - Sample & Assay Technologies
  21. 21. miScript miRNA Mimics act just like endogenous miRNA . Features of miScript miRNA Mimics: Double-stranded RNA oligonucleotide Functional sequence is the same as the natural mature miRNA Transfection results in inhibition comparable to the endogenous miRNA Stable in culture for up to 72 hours - 21 - Sample & Assay Technologies
  22. 22. Example Plate cDNA Sample 1 cDNA Sample 2 cDNA Sample 3 cDNA Sample 4 cDNA Sample 5 cDNA Sample 6 - 22 - Sample & Assay Technologies
  23. 23. Transcriptome PCR Array Results for INPPL1 - 23 - Sample & Assay Technologies
  24. 24. Site Predictions for INPPL1 TargetScan PicTar miRANDA miR-10 miR-184 miR-18a miR-135 miR-205 miR-18b miR-146 miR-342-3p miR-184 miR-642 miR-18ab miR-671-3p miR-205 miR-27ab miR-29abc - 24 - Sample & Assay Technologies
  25. 25. Site Predictions for INPPL1 TargetScan PicTar miRANDA miR-10 miR-184 miR-18a miR-135 miR-205 miR-18b miR-146 miR-342-3p miR-184 miR-642 miR-18ab miR-671-3p miR-205 miR-27ab miR-29abc - 25 - Sample & Assay Technologies
  26. 26. Compare Results with Research Community What’s really exciting…. Three new miRNAs that play a role in INPPL1 expression mir-193b mir-138 mir-373 - 26 - Confirm in your biological System Sample & Assay Technologies
  27. 27. Webinar Topics . Transcriptional Regulation and Mechanisms What regulates your gene’s expression? Techniques . What is a Transcriptome PCR Array? General Description Protocol Overview . Discovery in an Afternoon Application Example . Data Analysis and Technical Tips Easy to use Downloadable Excel Templates . What’s Next and Summary Follow-up Experiments - 27 - Sample & Assay Technologies
  28. 28. Data Analysis - 28 - Sample & Assay Technologies
  29. 29. Tips for Success Know your Gene Human Orthologue Validated qPCR Assay Either SYBR Green or Hydrolysis Probe Single-plex or Multiplex Expression Pattern Is GOI expressed in HeLa, HEK293, MCF…? . Normalization Use one or more housekeeping genes Data Analysis Spreadsheet Simplifies ∆∆Ct calculations . - 29 - Sample & Assay Technologies
  30. 30. Webinar Topics . Transcriptional Regulation and Mechanisms What regulates your gene’s expression? Techniques . What is a Transcriptome PCR Array? General Description Protocol Overview . Discovery in an Afternoon Application Example . Data Analysis and Technical Tips Easy to use Downloadable Excel Templates . What’s Next and Summary Follow-up Experiments - 30 - Sample & Assay Technologies
  31. 31. Now what?............. Suggestions to Validate your “Hits” miRNA-based Discovery Perform a miScript miRNA mimic experiment Identical Gain-Of-Function experiment Perform a miScript miRNA inhibitor experiment Loss-of-Function Experiment Peform a miScript Target Protector experiment ssRNA that disrupt a specific Target-miRNA interaction . - 31 - Sample & Assay Technologies
  32. 32. Now what?............. Suggestions to Validate your “Hits” miRNA-based Discovery Perform a miScript miRNA mimic experiment Identical Gain-Of-Function experiment Perform a miScript miRNA inhibitor experiment Loss-of-Function Experiment Peform a miScript Target Protector experiment ssRNA that disrupt a specific Target-miRNA interaction Pathway-based Discovery Repeat chemical treatment Dose curve or a Time Course Knockdown primary chemical inhibitor target Contact us -> pair your inhibitor with the correct reagent . . - 32 - Sample & Assay Technologies
  33. 33. Now what?............. Suggestions to Validate your “Hits” miRNA-based Discovery Perform a miScript miRNA mimic experiment Identical Gain-Of-Function experiment Perform a miScript miRNA inhibitor experiment Loss-of-Function Experiment Peform a miScript Target Protector experiment ssRNA that disrupt a specific Target-miRNA interaction Pathway-based Discovery Repeat chemical treatment Dose curve or a Time Course Knockdown primary chemical inhibitor target Contact us -> pair your inhibitor with the correct reagent Transcription Factor-based Discovery Repeat siRNA treatment Different siRNA design Rescue experiment – Expression vector with target sequence (QIAgenes) . . . - 33 - Sample & Assay Technologies
  34. 34. Product Information Product Contents Cat. No. SureFIND Transcriptome PCR Array One 96-well PCR plate (A or C format) 336611 Cells Treated With Cell Lines Treated Pathways miRNA mimetic HeLa Cancer Immunopathology Development & Differentiation Complete miRNome Drugs HEK293, HeLa, MCF-7 Chemical Inhibitors siRNAs MCF-7 Cancer-related Transcription Factors Expressed Transcription Factors - 34 - Sample & Assay Technologies
  35. 35. Where do I find out more? - 35 - Sample & Assay Technologies
  36. 36. List of Treatments - 36 - Sample & Assay Technologies
  37. 37. Summary Largest collection of cDNA panels derived from treated cells Choice One plate packs Multiple cell lines Drug treatments, miRNA mimics and siRNAs Included Data Analysis Spreadsheet Accelerating the Discovery Process - 37 - Sample & Assay Technologies
  38. 38. SureFIND Transcriptome PCR Array Pilot Study Offer Questions? Contact Technical Support 9 AM – 6 PM Eastern M – F Telephone: 888-503-3187; Email: support@SABiosciences.com Samuel Rulli, Ph.D.: Samuel.Rulli@qiagen.com - 38 - Sample & Assay Technologies

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