Last class

Plasmid isolation from bacteria

Paper 2: miRNAs in iPSCs

miRNAs

What are miRNAs?

Why are they important?

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?

Using computer algorithms for miRNA targets

Different algorithms to identify targets (Why?)Which one is best?

Common targets better?

So, what use are they?

Using miRNA target databases

Using miRNA target databases

Using miRNA target databases

Database will give you list

How will you pick target?

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

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?

How to design primers?

What do you need to know?

What are important considerations?

How to design primers?

How will you design primers?

Hard way:

Easy way:

Using Primer3Plus

OligoCalc

PrimerBLAST

Primers designed: Now what?

Enter in Google Drive Spreadsheet

http://tinyurl.com/m116lsp2015

DO NOT EDIT someone else’s already existing data!

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