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

2. Lab 8 Hard copy: Plasmid worksheets!

3. 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

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

5. The RT reaction What are components for RT reaction?

6. Controls RTPCR RT PCR RT PCR

7. Controls -RTPCR -RT PCR -RT PCR

8. 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

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

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

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

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

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

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

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

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

17. TemplatePrimerdNTPs Quantifying PCR products TemplatePrimerdNTPsProduct

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

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

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

21. 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

22. Other roles of RNAs What can you use complementarity for?