Techniques: RNA Flashcards
Is RNA extraction now kit based?
Yes
How do you do a gel electrophoresis?
Pores in gel separate RNA on mass. -ve RNA travels towards +ve anode.
How do you do a reverse transcription PCR?
1) Alter RNA into more stable cDNA using oligo dT primers to bind to the poly A tail
2) Normal PCR
How do you do an RT-PCR?
1) Dentature
2) Polymerisation
3) Signal Detection
All with SYBR
4) Annealing
5) Polymeric and strand displacement
6) Cleavage
7) Signal detection
All with TAQMAN
What makes an RT-PCR quantitative?
The fluorescence which increases with quantity of RNA
What is SYBR?
Binding dye you can see (might fail)
What is TaqMan?
Primer/Probe = As cDNA is copied probe is cleaved giving of fluorescence (Shouldn’t fail).
What is an RT-qPCR amplification plot?
Machine sets threshold and is a way to assess what RNA you have more off (as this will hit the threshold first). This also generates numbers
What is an RT-qPCR dissociation curve/melt curve?
This is used to see if primer is working correctly
Why would you use RNA interferance?
Get rid off RNA/Genes
How to do RNA interferance?
1) siRNA introduced into cell
2) siRNA binds to complementary targets within cell
3) RNA degraded
How to do in RNA in situ hybridisation?
1) Fix tissue to preserve RNA structure
2) Make tissue permeable
3) Denature target RNA
4) Hybridise probe and RNA
5) Fluorescence microscopy to see probe
6) Visualise location of hybridised probe to determine location of target RNA within the tissue
