1. Last class Plasmid isolation from bacteria Paper 2: miRNAs in iPSCs

2. miRNAs What are miRNAs? Why are they important?

3. Doing real science! miR- NNN is involved in cancers What might we want to know? How to identify miR- NNN targets? - Does target need to be completely complementary? - Are all complementary sequences targets? How to identify miR-23b targets?

4. Using computer algorithms for miRNA targets Different algorithms to identify targets (Why?) Which one is best? Common targets better? So, what use are they?

5. Using miRNA target databases

7. Database will give you list How will you pick target?

8. 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

9. After picking a target… Pick a target – then what? How will you “validate” target? What controls do you need to include? DNA contamination? Amount of sample? Change in levels due to miRNA targeting?

10. How to design primers? What do you need to know? What are important considerations?

11. How to design primers? How will you design primers? Hard way: Easy way:

12. Using Primer3Plus

13. OligoCalc

14. PrimerBLAST

15. Primers designed: Now what? Enter in Google Drive Spreadsheet http://tinyurl.com/m116lsp2015 DO NOT EDIT someone else’s already existing data! Lab Section Group number Group members Transcript Gene Reason