QIAGEN microRNA Seminar


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This presentation gives an overview on how new tools from QIAGEN facilitate miRNA research, from purification through to functional analysis.

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QIAGEN microRNA Seminar

  1. 1. Facilitating miRNA research – from purification to functional analysis <ul><li>The applications presented here are for research purposes. Not for use in diagnostic procedures. </li></ul>
  2. 2. microRNAs - micromanagers of gene expression <ul><ul><li>Characteristic of miRNAs </li></ul></ul><ul><ul><ul><li>Naturally occurring, endogenous small RNA </li></ul></ul></ul><ul><ul><ul><li>Regulate at least 1/3 of the protein encoding genes by binding to the mRNA </li></ul></ul></ul><ul><ul><ul><li>> 1200 miRNAs in human (miRBase V15.0) </li></ul></ul></ul><ul><ul><ul><li>A mature miRNA is approximately 20 - 22 nt long </li></ul></ul></ul><ul><ul><ul><li>miRNA seed sequences (2-7 nt) are crucial in target selection </li></ul></ul></ul><ul><ul><ul><li>Imperfectly base pair to the target and result in translational repression </li></ul></ul></ul><ul><ul><ul><li>A given miRNA may have multiple target transcripts </li></ul></ul></ul><ul><ul><li>Target mRNAs </li></ul></ul><ul><ul><ul><li>miRNA binding sites are typically at the 3’-UTR of the target transcripts </li></ul></ul></ul><ul><ul><ul><li>Typically multiple miRNA binding sites are present for a given transcript </li></ul></ul></ul>Ω Seed region 5’ 3’ miRNA Target mRNA 5’ 3’ Ω miRNA 5’ 3’
  3. 3. Biogenesis of microRNA Pri-miRNA= Primary microRNA Pre-miRNA = Precursor microRNA
  4. 4. QIAGEN solutions for microRNA research miRNeasy Kits 1 miScript PCR System 2 miScript RT 2 miRNA PCR Arrays 3 miScript miRNA Mimics and Inhibitors 4 miScript Target Protector 5
  5. 5. <ul><ul><li>Efficient purification of miRNA from different starting materials </li></ul></ul><ul><ul><ul><li>Tissue </li></ul></ul></ul><ul><ul><ul><li>Cells </li></ul></ul></ul><ul><ul><ul><li>Blood </li></ul></ul></ul><ul><ul><ul><li>Serum/Plasma </li></ul></ul></ul>
  6. 6. miRNeasy - copurification or enrichment of miRNA <ul><li>Flexible protocols: </li></ul><ul><ul><li>Copurification of miRNA & total RNA </li></ul></ul><ul><ul><li>miRNA enriched fraction </li></ul></ul><ul><li>Formats: </li></ul><ul><ul><li>Single spin </li></ul></ul><ul><ul><li>96-well plate </li></ul></ul><ul><ul><li>Manual or automated on QIAcube </li></ul></ul><ul><li>Principle: </li></ul><ul><ul><li>Proven silica membrane technology </li></ul></ul>
  7. 7. miRNA isolation from different starting material Human Animal Blood Serum/plasma Species Sample specification Kit Cells Tissue  unfixed  FFPE fixed sections  fixed in PAXgene containers  miRNA  miRNA + protein Blood Serum/plasma Cells Tissue  unfixed  FFPE fixed sections  fixed in PAXgene containers  miRNA  miRNA + protein  miRNeasy Protect Animal Blood Kit  miRNeasy Mini Kit (UDP)  PAXgene Blood miRNA Kit  QIAamp Circulating Nucleic Acid Kit (SP) <ul><li>miRNeasy Mini/96 Kit </li></ul><ul><li>AllPrep® DNA/RNA/Protein Mini Kit* </li></ul> miRNeasy Mini/96Kit  miRNeasy FFPE Kit  PAXgene Tissue miRNA Kit <ul><li>miRNeasy Kit </li></ul><ul><li>AllPrep® DNA/RNA/Protein Mini Kit* </li></ul>Plant <ul><li>RNeasy Lipid Tissue Mini Kit* (UDP) </li></ul><ul><li>RNeasy Mini Kit* (UDP) </li></ul> miRNeasy Mini/96Kit  miRNeasy FFPE Kit  PAXgene Tissue miRNA Kit * additionally needed RNeasy MinElute ® Cleanup Kit; UDP = User Developped Protocol; SP = Supplementary Protocol Virus <ul><li>depending on virus </li></ul>
  8. 8. <ul><ul><li>Detection - The miScript PCR System miRNA quantification by real-time PCR </li></ul></ul><ul><ul><ul><li>mature microRNA </li></ul></ul></ul><ul><ul><ul><li>precursor microRNA </li></ul></ul></ul><ul><ul><ul><li>other small non-coding RNAs </li></ul></ul></ul><ul><ul><ul><li>mRNAs </li></ul></ul></ul>
  9. 9. miScript PCR System All from the same cDNA
  10. 10. miScript PCR System Reverse transcription - principle
  11. 11. miScript RT Protocol: One-step reaction
  12. 12. miScript PCR System Real-time PCR detection of mature miRNAs mRNA miRNA
  13. 13. miScript RT System Linear cDNA synthesis from 10 pg – 1µg RNA 1 µg to 10 p g of input RNA from HeLa S3 cells, used for miScript RT reaction in 20 µl volume. 2µl of this cDNA (100 n g to 1 pg) was used in PCR 2 cells 2x10 5 cells Detection to <20 copies
  14. 14. Specificity of miScript Primer Assays: Let-7 Isoforms Human Let-7 Isoforms Cross-reactivity
  15. 15. <ul><ul><li>The miScript PCR System </li></ul></ul><ul><ul><ul><li>miScript Control Assays </li></ul></ul></ul>
  16. 16. miScript Control Assays
  17. 17. miScript Control Assays: Constant level of expression across cell types  Constant level of expression across cell types  Similar small size as miRNA  A wide range of expression level to choose the most suitable control  Cross species
  18. 18. <ul><ul><li>microRNA expression profiling </li></ul></ul><ul><ul><ul><li>RT 2 miRNA PCR Arrays </li></ul></ul></ul>
  19. 19. RT 2 miRNA PCR Array System microRNA profiling <ul><li>RT 2 miRNA First Strand Kit </li></ul><ul><ul><li>Preferentially reverse transcribe mature miRNA </li></ul></ul><ul><ul><li>Built-in external RNA control </li></ul></ul><ul><ul><li>One-step reaction </li></ul></ul><ul><li>PCR Arrays: pathways and genomes </li></ul><ul><ul><li>Cancer </li></ul></ul><ul><ul><li>Cell Differentiation & Development </li></ul></ul><ul><ul><li>Immunopathology </li></ul></ul><ul><ul><li>Inflammation </li></ul></ul><ul><ul><li>miFinder </li></ul></ul><ul><ul><li>Whole Genome (miRNome) – Sanger miRBase V14.0 </li></ul></ul><ul><li>Optimized RT 2 SYBR ® Green Mastermix </li></ul><ul><li>RT 2 miRNA Data Analysis Excel Template & Web Portal </li></ul>
  20. 20. How RT 2 miRNA PCR Arrays work <ul><li>cDNA synthesis </li></ul><ul><ul><li>2 hours </li></ul></ul><ul><li>Load plates </li></ul><ul><ul><li>2 minutes </li></ul></ul><ul><li>Run 40 cycle qPCR program </li></ul><ul><ul><li>2 hours </li></ul></ul><ul><li>Upload and analyze data </li></ul><ul><ul><li>15 minutes </li></ul></ul>Compatible with many cyclers
  21. 21. Anatomy of a miRNA PCR Array Each plate contains: 4 housekeeping snoRNA genes 4 quality controls (2 reverse transcription & 2 PCR controls) Available for human, mouse, and rat
  22. 22. Data Analysis <ul><li>Web-based software </li></ul><ul><ul><li>No installation needed </li></ul></ul><ul><li>From raw Ct values to fold change results </li></ul><ul><ul><li>Using ∆∆C(t) Method </li></ul></ul><ul><li>Multiple analysis formats </li></ul><ul><ul><li>Scatter plot </li></ul></ul><ul><ul><li>Volcano plot </li></ul></ul><ul><ul><li>Multi-group plot </li></ul></ul><ul><ul><li>Clustergram </li></ul></ul>Also available as downloadable Excel templates Clustergram
  23. 23. Volcano Plot
  24. 24. Scatter Plot
  25. 25. Human Cancer miRNA RT 2 Array: Colon Cancer Cancer-specific miRNA upregulated in colon tumor. Are there specific miRNA expression differences in tumor versus normal adjacent tissue?
  26. 26. Application Data: Cell Differentiation & Development Human Cell Differentiation & Development RT 2 miRNA PCR Array identifies 26 brain- and 20 muscle-specific miRNA potentially regulating tissue-specific gene expression. 26 Brain-Specific miRNA 20 Muscle-Specific miRNA
  27. 27. <ul><ul><li>microRNA functional analysis </li></ul></ul><ul><ul><ul><li>miScript miRNA Mimics and Inhibitors </li></ul></ul></ul><ul><ul><ul><li>miScript Target Protectors ( New ) </li></ul></ul></ul>
  28. 28. Tools to study miRNA functions <ul><li>Synthetic RNA duplex </li></ul><ul><li>Mimics the effect of an endogenous miRNA </li></ul><ul><li>Single stranded modified RNA </li></ul><ul><li>Targets of miRNA should increase when miRNA is inhibited </li></ul>
  29. 29. miScript Target Protector Principle Target mRNA A Target mRNA B Target mRNA C    one and the same miRNA can bind to several targets  no target is expressed (  )    Inhibitor binds to miRNA  each target is expressed (  )    miScript Target Protector binds only to one specific target  specific target is expressed while other targets are not expressed
  30. 30. miScript Target Protector Protection of a luciferase reporter against endogenously expressed miR-16 <ul><ul><li>Verification of miRNA binding site </li></ul></ul><ul><ul><li>Reliable, reproducible miRNA inhibition after transfection </li></ul></ul><ul><li>of miScript Target Protector miR-16 </li></ul>RLU
  31. 31. miScript Target Protector miR-9 causes down regulation of an important gene for cell viability <ul><ul><li>Determination of miRNA role in a pathway or process </li></ul></ul><ul><ul><li>miScript Target Protector miR-9 inhibits the down regulation by miR-9 </li></ul></ul><ul><ul><li>allowing the cell viability gene to be expressed </li></ul></ul>Untreated cells Cell death caused by miR-9 Cell survival after treatment with TP TP = Target Protector
  32. 32. miScript Target Protector Target Specificity of the Target Protector <ul><ul><li>Transfection of miR-1 mimic leads to down-regulation of different miR-1 targets including ADAR and HDAC4 </li></ul></ul><ul><ul><li>After cotransfection of a target protector designed against the miR-1 MRE in ADAR, ADAR protein was no longer downregulated by a miR-1 mimic </li></ul></ul><ul><ul><li>HDAC4 downregulation was not affected by the presence of the ADAR target protector </li></ul></ul>miR-1 mimic was co-transfected in all samples except UT 55 kD 130 kD ADAR TUBB HDAC4 TUBB UT 1 Neg ctrl 0.1 0.5 0.05 ADAR Target Protector [µM] UT 1 Neg ctrl 0.1 0.5 0.05 ADAR Target Protector [µM]
  33. 33. Typical workflow miRNA research A complete offering for miRNA research! miScript Precursor Assay miScript PCR Starter Kit Rotor-Gene Q QIAgility PAXgene Tissue miRNA Kit PAXgene Blood miRNA Kit QIAamp Circulating Nucleic Acid Kit miRNeasy Protect Animal Blood Kit miScript PCR Control Set QIAcube miScript Target Protector Mimic and Inhibitor Sets miScript RT2 miRNA PCR Arrays miScript PCR Control
  34. 34. Thank you!