qRT-PCR help! - (Sep/26/2007 )
I am new to qRT-PCR, although I have done quantitative real time PCRs a lot. My question is if you start with the same amount of RNA for all your reverse transcription reactions, and you assume that the RT reaction is 100% efficient, do you still quantitate the cDNA to use for the real time PCR? And if you do quantitate the cDNA, aren't you creating room for error, because its the RNA that's supposed to be detected and not the cDNA? As you can see, I am obviously confused Any advice would be highly appreciated!
Answering to your question, no, it's not necessary or even recomended to quantify the cDNA. However, because it is not very safe to assume that the RT reaction efficiency is always going to be 100%, you should correct for that by analysing some housekeeping gene(s) cDNA levels in the same sample and using that to correct your data.
Thanks Erica! Thats what I had thought..but I dont have primers for a housekeeping gene in my sample. However since I am doing absolute quantification, I shall be using a standard curve of known dilutions, does that make sense?