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Mi rna functional analysis 2013
Mi rna functional analysis 2013
Mi rna functional analysis 2013
Mi rna functional analysis 2013
Mi rna functional analysis 2013
Mi rna functional analysis 2013
Mi rna functional analysis 2013
Mi rna functional analysis 2013
Mi rna functional analysis 2013
Mi rna functional analysis 2013
Mi rna functional analysis 2013
Mi rna functional analysis 2013
Mi rna functional analysis 2013
Mi rna functional analysis 2013
Mi rna functional analysis 2013
Mi rna functional analysis 2013
Mi rna functional analysis 2013
Mi rna functional analysis 2013
Mi rna functional analysis 2013
Mi rna functional analysis 2013
Mi rna functional analysis 2013
Mi rna functional analysis 2013
Mi rna functional analysis 2013
Mi rna functional analysis 2013
Mi rna functional analysis 2013
Mi rna functional analysis 2013
Mi rna functional analysis 2013
Mi rna functional analysis 2013
Mi rna functional analysis 2013
Mi rna functional analysis 2013
Mi rna functional analysis 2013
Mi rna functional analysis 2013
Mi rna functional analysis 2013
Mi rna functional analysis 2013
Mi rna functional analysis 2013
Mi rna functional analysis 2013
Mi rna functional analysis 2013
Mi rna functional analysis 2013
Mi rna functional analysis 2013
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Mi rna functional analysis 2013

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  • 1. Functional analysis of miRNA Samuel J. Rulli, Jr. Ph.D. samuel.rulli@QIAGEN.com Sample & Assay Technologies
  • 2. Topics for webinar What is microRNA? Manipulation of miRNA in cell lines using reporter assays miRNA Mimics Inhibitors Target protectors Screening your target gene for miRNA regulation Ready-to-use cDNA panels Identification of miRNA regulation by qPCR miRNA Targets PCR Arrays Summary Q & A session -2- Sample & Assay Technologies
  • 3. What Is Micro RNA? RISC Target mRNA 5’ CDS 3’ 3’ 5’ miRNA Seed region Endogenously expressed small functional RNAs (~ 21nt -24 nt) Regulate mRNA expression post-transcriptionally Involves the formation of RISC (RNA induced Silencing Complex) Translational Repression and or mRNA degradation Only partial sequence complementarity required A given miRNA can regulate multiple mRNAs A given mRNA can be regulated by multiple miRNAs Another layer of complexity for regulating gene expression Aberrant expression of miRNAs is associated with many diseases e.g. Many different Cancers -3- Sample & Assay Technologies
  • 4. Canonical pathway of microRNA 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 -4- Sample & Assay Technologies
  • 5. A growing picture of miRNA dysregulation in cancer, developmental and metabolic pathways In the last 5 years, a substantial number of studies and reviews have associated the presence of various miRNAs with cell proliferation, resistance to apoptosis, and differentiation in cancer cells. . . . For example: . Deletions of miRNA-regulated genes have been detected in more than 65% of chronic lymphocytic leukemia cases, in 50% of mantle-cell lymphomas, in 16% to 40% of multiple myelomas, and in 60% of prostate cancers. Other miRNA abnormalities have been reported in a wide variety of Human neoplasms, including other hematologic malignancies such as promyelocytic leukemia Benign tumors such as leiomyoma and pituitary adenoma Multiple types of carcinomas, including pancreatic, esophageal, thyroid, lung, and breast Neuroblastomas and glioblastomas -5- Sample & Assay Technologies
  • 6. Topics for webinar What is microRNA? Manipulation of miRNA in cell lines using reporter assays miRNA Mimics Inhibitors Target protectors Screening your target gene for miRNA regulation Ready-to-use cDNA panels Identification of miRNA regulation by qPCR miRNA Targets PCR Arrays Summary Q & A session -6- Sample & Assay Technologies
  • 7. Manipulation of miRNA in cell lines Functional interactions between miRNA and mRNA can be follow using: Reporter assays: reporter gene has miRNA site in 3’ UTR which decreases reporter gene translation or causes transcriptional degradation Real-time PCR: monitors transcriptional degradation of mRNA Western blot: monitors transcriptional or translational changes Pathway reporter assay: monitors the pathway activity of specific transcription Factors. -7- Sample & Assay Technologies
  • 8. miRNA Mimics & Inhibitors: How do they work? Transfection of Mimics Transfection of Inhibitors RISC Target Expression Level -8- Sample & Assay Technologies
  • 9. miRNA Mimics & Inhibitors: How do they work? Transfection of Mimics Transfection of Inhibitors Mimics increase miRNA activity RISC Target Expression Level -9- Sample & Assay Technologies
  • 10. Comparable down-regulation by mimics and endogenous miRNAs Hela cells with reporter assays for miR-1 or miR-16. miR-16 is endogenously Expressed, while miR-1 was introduced using a miRNA mimic - 10 - Sample & Assay Technologies
  • 11. Stability of miRNA mimics miRNA mimics are stable for at least 72 hours. - 11 - Sample & Assay Technologies
  • 12. HDAC4 is a target of mir-1 HDAC4 HDAC4 had 2 miR-1 interaction sites. HeLa cells transfected with mir-1 show down Regulation of HDAC4 at the mRNA and protein levels. Sample & Assay Technologies
  • 13. miRNA Mimics & Inhibitors: How do they work? Transfection of Mimics Transfection of Inhibitors Inhibitor decrease miRNA activity RISC Target Expression Level - 13 - Sample & Assay Technologies
  • 14. mir-16 is decreased by miR-16 inhibitor in HeLa cells by reporter assay - 14 - Sample & Assay Technologies
  • 15. Application Example: miRNA & Pathway Activity The let-7 family of miRNAs is widely viewed as playing a role in tumor suppression. . Expression of let-7 family members is downregulated in many cancer types and during tumor progression in comparison to normal tissue. . Humans have 10 mature let-7 family sequences (let-7a, 7b, 7c, 7d, 7e, 7f, 7g, 7i, miR-98, and miR-202) . Interestingly, certain let-7 species are also known to be upregulated in some cancer types. These data on the expression of let-7 in various cancers indicate that individual let-7 family members can have different activities. . Question: Which let-7 family members effect cell cycle progression and how? . - 15 - Sample & Assay Technologies
  • 16. Cignal Reporters: What You Need • Cignal Cell Cycle Pathway Reporter Measures E2F/DP1 Activity • Transfection of HEK293 cells • Biological Test •Let-7 family mimics • Dual-Luciferase Assay - 16 - Sample & Assay Technologies
  • 17. Cignal Reporter Assays (45 Pathways) Pathway Amino Acid Deprivation Androgen Antioxidant Response ATF6 C/EBP cAMP/PKA Cell Cycle DNA Damage EGR1 ER Stress Estrogen GATA Glucocorticoid Heat Shock Response Heavy Metal Stress Hedgehog HNF4 Hypoxia Interferon Regulation Type I Interferon Interferon Gamma KLF4 Liver X Transcription Factor ATF4/ATF3/ATF2 Androgen Receptor Nrf2 & Nrf1 ATF6 C/EBP CREB E2F/DP1 p53 EGR1 CBF/NF-Y/YY1 ER GATA GR HSF MTF1 GLI HNF4 HIF-1 IRF-1 STAT1/STAT2 STAT1/STAT1 KLF4 LXR - 17 - Pathway MAPK/ERK MAPK/JNK MEF2 c-myc Nanog NFκB Notch Oct4 Pax6 PI3K/AKT PKC/Ca++ PPAR Progesterone Retinoic Acid Retinoid X Sox2 SP1 STAT3 TGFβ Vitamin D Wnt Xenobiotic Transcription Factor Elk-1/SRF AP-1 MEF2 Myc/Max Nanog NFκB RBP-Jk Oct4 Pax6 FOXO NFAT PPAR PR RAR RXR Sox2 SP1 STAT3 SMAD2/3/4 Vitamin D Receptor TCF/LEF AhR Sample & Assay Technologies
  • 18. Let-7 Family Members and the Cell Cycle - 18 - Sample & Assay Technologies
  • 19. Topics for webinar What is microRNA? Manipulation of miRNA in cell lines using reporter assays miRNA Mimics Inhibitors Target protectors Screening your target gene for miRNA regulation Ready-to-use cDNA panels Identification of miRNA regulation by qPCR miRNA Targets PCR Arrays Summary Q & A session - 19 - Sample & Assay Technologies
  • 20. Approach to Identifying miRNA Regulators miRNA mimic miRNA 1 2 3 4 1) Treat Cells 2) Isolate RNA 3) First Strand cDNA Synthesis 4) qPCR for Gene Expression - 20 - Sample & Assay Technologies
  • 21. Transcriptome cDNA panels Transcriptome PCR Arrays are ready-to-use cDNA panels for discovering the miRNAs, 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 miRNA mimic treatment. . - 21 - Sample & Assay Technologies
  • 22. Example Transcriptome PCR Array cDNA Sample 1 cDNA Sample 2 cDNA Sample 3 cDNA Sample 4 cDNA Sample 5 cDNA Sample 6 - 22 - Sample & Assay Technologies
  • 23. Materials needed for miRNA discovery . . . Transcriptome PCR Array (1 for each GOI or HKG) The Research Also Needs: qPCR Thermocycler Micropipettor qPCR Master Mix qPCR Assay (either SYBR® green or Probe-based Assays) Computer with Microsoft Excel for Data Analysis - 23 - Sample & Assay Technologies
  • 24. SureFIND Protocol - 24 - Sample & Assay Technologies
  • 25. miRNA pathways available • Cancer miRNAs • Immunopathology • Development and differentiation • Complete miRNome (~900 miRNAs) Sample & Assay Technologies
  • 26. 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 . - 26 - Sample & Assay Technologies
  • 27. 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 - 27 - Sample & Assay Technologies
  • 28. Cancer miRNA Transcriptome PCR Array Results for INPPL1 - 28 - Sample & Assay Technologies
  • 29. 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 - 29 - Sample & Assay Technologies
  • 30. 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 - 30 - Sample & Assay Technologies
  • 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. Topics for webinar What is microRNA? Manipulation of miRNA in cell lines using reporter assays miRNA Mimics Inhibitors Target protectors Screening your target gene for miRNA regulation Ready-to-use cDNA panels Identification of miRNA regulation by qPCR miRNA Targets PCR Arrays Summary Q & A session - 32 - Sample & Assay Technologies
  • 33. Identification of transcriptional degradation using qPCR: RT2 Profiler microRNA Targets PCR Arrays Transfection of Mimics Transfection of Inhibitors RISC Target Expression Level - 33 - Sample & Assay Technologies
  • 34. Anatomy of a RT2 Profiler microRNA Targets PCR Arrays miR-21 Targets PCR Array • 84 Experimentally verified or predicted targets of miRNA • 5 Housekeeping Genes • Genomic DNA Contamination Control • Reverse Transcription Controls (RTC) n=3 • Positive PCR Controls (PPC) n=3 Supported PCR Array Formats • • • • 96-Well Plate 384-Well Plate 100-Well Disc 96x96 Chip - 34 - Sample & Assay Technologies
  • 35. Available RT² Profiler microRNA Targets PCR Arrays: Human miR-21 miR-155 Mouse miR-21 miR-155 Rat miR-21 miR-17 & miR-20a miR-17 & miR-20a miR-17 & miR-20a miR-146a miR-146a miR-146a miR-1 & miR-206 miR-1a & miR-206 miR-1 & miR-206 miR-34a miR-34a miR-34a miR-221 & miR-222 miR-221 & miR-222 miR-221 & miR-222 let-7 let-7 let-7 Custom miRNA targets arrays are possible based on your miRNA of interest - 35 - Sample & Assay Technologies
  • 36. How RT2 Profiler PCR Arrays Work cDNA Synthesis (C-03 kit) – Genomic DNA Removal Step (5 min.) – Reverse Transcription Step (20 min.) . Load Plates 1 Sample per PCR Array 2 minutes with multi-channel pipet . Run 40 cycle qPCR Program Standard cycling conditions for all Real Time PCR Instruments 2 hours . Upload and Analyze Data 15 minutes from Raw Ct to Fold Change Data . - 36 - Sample & Assay Technologies
  • 37. Clustergram Data . . . Analysis Web-Based Software Scatter Plot No installation or licensing needed From Raw Ct Values to Fold Change Results ∆∆C(t) Method Multiple Analysis Formats Scatter Plot Volcano Plot Multi-Group Plot Clustergram Volcano Plot Also available as downloadable Excel templates - 37 - Sample & Assay Technologies
  • 38. Summary Global miRNA function can by manipulated by using mimics and inhibitors Specific miRNA/mRNA interactions can be studied using target protectors Pathway or transcription factor assays can be used to follow miRNA affects on On cell biology RT2 miRNA Targets PCR Arrays facilitate screening miRNA target genes using verified qPCR Assays Sample & Assay Technologies
  • 39. Questions, Comments, Concern or Suggestions? Answers to your technical and biological questions: Technical Support: 888-503-3187 support@sabiosciences.com Samuel Rulli Samuel.Rulli@QIAGEN.com Copy of slides? Type slides into questions box or fill out survey on exit Want someone to call you? Type contact me into questions box Want to try something? Request demos in the questions box. miRNA mimics, inhibitors or target protectors Cignal reporter assays cDNA panels miRNA Targets RT2 Profiler PCR Arrays - 39 - Sample & Assay Technologies

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