New to rt pcr real time - guidelines and advice (Nov/23/2007 )
I'm brand new to real time rt -pcr (and tp pcr itself!) but i guess i'm not alone on this. I'd like to learn the basics about it and get started. To all you "newbies"...welcome...
Anyway, here's my first question: which parameter is the most important when trying to determine results (amplification or not amplification) if i dont know initial RNA quantity?
Is it Ct, efficiency, etc.?
Questions, sugestions and critics are all welcome here. Once again, Thank you.
Well, the best is get started. Once you do couple of runs, you will know lots of things. By the way do some home-work on primer design and recipes as well. good luk!
to make a long story short, look for these three parameters:
1. linear standard curve (r²>0.980)
2. consistency across replicates (Cts between your replicates should vary < +/- 0.3)
and 3. high amplification efficiency (90-105%)
if these 3 points apply to your assays, you should get good qPCR data
Thanks for your posts guys!!!
Well i must say i've alredy done a couple of runs using 3 primers from literature and 1 designed by a teacher. Not so difficult when it all comes in a kit!!!!
However, performing a technique is not an issue when there's some knowledge about basic facts and following recipes (which i've gathered like crazy). Problem comes when trying to interpretate results.
I realize those three parameters are quite important but i really need some more specific directions...i.e, how do i calculate efficiency? Which one is considered a good Ct?
forgive my ignorance but i'm quite confussed about parameters and interpretation, by the way i'm not working with replicates...he he...
Thank you all!