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

2. Use qPCR to quantify expression level
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? ? ?

4. 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

5. Lab session Group B Group A miRNA Control Control RNA miRNA RNA

6. Quantifying DNA/RNA: qPCR
Start with 1 molecule
Start with 10 molecules
1 cycle
2 cycles
3 cycles
30 cycles
What would happen with 1000 vs 10,000 molecules?

7. EtBr Gels…
PCR data: BEST conclusion?

8. EtBr Gels (continued)…
PCR data: BEST conclusion?

9. Solution: qPCR

10. “Threshold” cycle

11. “Threshold” cycle

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

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

14. After picking a target…
What controls do you need to include?
Transfection / RNA introduction affected transcription
DNA contamination?
Amount of sample ? Endogenous expression?
Change in levels due to miRNA targeting?

15. Controls RT RT RT
PCR
PCR
PCR
Product
Product
Product

16. Controls
-RT
-RT
-RT
PCR
PCR
PCR
Product
Product

17. qPCR – Things to think about…
qPCR data: Conclusion? 1 2 3
Fluorescence units
Wildtype
miR-23b overexpression

18. After picking a target…
What controls do you need to include?

19. qPCR – Things to think about…
qPCR data: Conclusion? 1 2 3
Normalized fluorescence
Wildtype
miR-23b overexpression

20. Controls Transfection / RNA introduction affected transcription
DNA contamination?
Amount of sample ? Endogenous expression?
Change in levels due to miRNA targeting?

21. “Threshold” cycle

22. 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

23. Use qPCR to quantify expression level
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