real time-pcr of cDNA samples - are the results reliable? (May/28/2008 )
I have a question about real time-PCR of cDNA.
Here's what I'm thinking: cDNA is a result of reverse transcription. Let's say there are 5 RNA samples. 1ug of each was used for reverse transcription. Unless we know how much of our target mRNA had been reverse transcribed properly and completely from each sample, there is no way the results of a real time-PCR on those cDNA samples can be reliable.
True or false?
Unless you're primers, for the real-time PCR, amplify the very end of the 5' side of the mRNA, I think you're o.k.
If you reverse transcribe with oligo dT then you might miss the 5' side of the mRNA.
You can get around it by using your 3' reverse primer for the RT or using random hexsamers for the RT.
In any case, my feeling is that the real-time method has been in use for long enough and that it is well established, so you can use it without fear.
If you don't believe in your results you can always verify them with a northern blot.