1. miRNA targets Using undergraduate molecular biology labs to discover targets of miRNAs in humans Adam Idica, Jordan Thompson, Irene Munk Pedersen, Pavan Kadandale

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 ? Lyse cells BindWashElute ??

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

5. Controls Product RTPCR RT PCR RT PCR

6. Controls Product -RTPCR -RT PCR -RT PCR

7. Quantifying DNA/RNA: qPCR 1 cycle 2 cycles 3 cycles 30 cycles Start with 1 molecule 2 molecules 4 molecules 8 molecules ~1 billion molecules Start with 10 molecules 20 molecules 40 molecules 80 molecules ~10 billion molecules

8. Same starting material… Looks different!

9. Different starting material… Looks the same!

10. Solution: qPCR

11. “Threshold” cycle

13. How can we quantify DNA in PCR? DNA molecules in PCR - Template - Primer - dNTPs - Product How to quantify ONLY product?

14. TemplatePrimerdNTPs Quantifying PCR products TemplatePrimerdNTPsProduct

15. qPCR – Things to think about… qPCR data: Conclusion? B. “X” is not target A. “X” is target C. Need a control 1 2 3 Fluorescence units Wildtype miR-128 overexpression

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

17. qPCR – Things to think about… qPCR data: Conclusion? B. “X” is not target A. “X” is target C. Need a control 1 2 3 Normalized fluorescence Wildtype miR-128 overexpression

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

19. “Threshold” cycle

20. Calculations  C T =  C T (control) -  C T (miR)  C T (miR) = C T (target-miR) - C T (endogenous control-miR)  C T (control) = C T (target-control) - C T (endogenous control-control) Expression fold change = 2  C T