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. We have primers: Now what? Pick target Design primers Isolate RNA from cells

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

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

6. RT-PCR What are components for RT-PCR?

7. Controls Product RTPCR RT PCR RT PCR

8. Controls Product -RTPCR -RT PCR -RT PCR

9. Lab session Each group will start with either miRNA or Control Make total RNA (freeze half!) Remaining: Split into TWO tubes Keep one, swap one with another group Make cDNA

10. Lab session Group A Control Control RNA Group B miRNA miRNA RNA Control RNAmiRNA RNAControl RNAmiRNA RNA

11. 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

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

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

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

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

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

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

18. TemplatePrimerdNTPs Quantifying PCR products TemplatePrimerdNTPsProduct

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

20. qPCR controls Normalize for overexpression of miR-23b? Positive control

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

22. 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

23. 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

24. miRNAs in disease How might miRs be involved in cancer? Role of miRs in treating cancer?