last class plasmid isolation from bacteria paper 2: mirnas in ipscs
TRANSCRIPT
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Last class
Plasmid isolation from bacteria
Paper 2: miRNAs in iPSCs
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miRNAs
What are miRNAs?
Why are they important?
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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?
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Using computer algorithms for miRNA targets
Different algorithms to identify targets (Why?)Which one is best?
Common targets better?
So, what use are they?
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Using miRNA target databases
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Using miRNA target databases
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Using miRNA target databases
Database will give you list
How will you pick target?
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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
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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?
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How to design primers?
What do you need to know?
What are important considerations?
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How to design primers?
How will you design primers?
Hard way:
Easy way:
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Using Primer3Plus
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OligoCalc
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PrimerBLAST
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Primers designed: Now what?
Enter in Google Drive Spreadsheet
http://tinyurl.com/m116lsp2015
DO NOT EDIT someone else’s already existing data!
Lab SectionGroup numberGroup membersTranscriptGene GeneReason