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Selecting Candidate miRNA Targets: A Step-by-Step Guide for Labs 7-9

This guide outlines the overall goal for Labs 7-9 on how to pick candidate miRNA targets and validate target predictions. The process involves selecting a target, designing primers, isolating RNA from cells, and synthesizing cDNA using RT-PCR. Using qPCR, you will quantify expression levels of your targets. Key steps include proper RNA isolation, cDNA creation, and quantifying results with qPCR controls. You'll explore different starting materials and normalization techniques for accurate expression level determination, ensuring robust experimental outcomes.

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Selecting Candidate miRNA Targets: A Step-by-Step Guide for Labs 7-9

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  1. Last class Overall goal for labs 7-9 How to pick candidate miRNA targets Overview: “Validate” target prediction

  2. Labs 7-9 flow chart Pick target Design primers Isolate RNA from cells Make cDNA using RT-PCR Use qPCR to quantify expression level Repeat

  3. RNA Isolation: Overview ? ? ? http://www.thermoscientificbio.com/uploadedImages/Products/Nucleic_Acid_Purification/Kits_-_Genomics/D_RNA_cleanup_and_concentration_protocol.jpg

  4. Labs 7-9 flow chart Pick target Design primers Isolate RNA from cells Make cDNA using RT-PCR

  5. RT-PCR What are components for RT-PCR? - RNA template - Primer: which? - dNTPs - RT - Buffer

  6. Controls RT RT RT PCR PCR PCR Product Product Product

  7. Lab session Each group will start with either miRNA or Control Make total RNA Make cDNA Make split into TWO tubes Keep one, swap one with another group

  8. Lab session Group B Group A miRNA Control miRNA cDNA Control cDNA Control cDNA miRNA cDNA Control cDNA miRNA cDNA

  9. Quantifying DNA/RNA: qPCR Start with 1 molecule Start with 10 molecules 1 cycle 2 cycles 3 cycles 30 cycles

  10. Same starting material… http://www.sabiosciences.com

  11. Different starting material… http://www.sabiosciences.com

  12. Solution: qPCR http://www.abbottmolecular.com

  13. “Threshold” cycle http://www.sabiosciences.com

  14. “Threshold” cycle http://www.sabiosciences.com

  15. How can we quantify DNA in PCR? DNA molecules in PCR How to quantify ONLY product?

  16. Quantifying PCR products Template Template Primer Primer dNTPs dNTPs Product Product

  17. qPCR controls Normalize amount of starting material? Could normalize total RNA Better method?

  18. qPCR controls Normalize for overexpression of miR-128? Positive control

  19. “Threshold” cycle http://www.sabiosciences.com

  20. Calculations DDCT = DCT(control) - DCT(miR) DCT(control) = CT(target-control) - CT(endogenous control-control) DCT(miR) = CT(target-miR) - CT(endogenous control-miR) Expression fold change = 2DDCT

  21. Labs 7-9 flow chart Pick target Design primers Isolate RNA from cells Make cDNA using RT-PCR Use qPCR to quantify expression level Repeat

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