RT-PCR primers and the poly(A) tail - (Jan/04/2012 )
I've been using a pair of qRT-PCR primers for a while with good results.
Suddenly somebody told me that the primers musn't be too far from the poly(A) tail of my mRNA if I use oligo d(T) as the priming method in my RT reaction, because "the RT reaction never works 100%".
This somebody claims she always keeps a distance of less than 1,000 bp between the beginning of the forward primer and the beginning of the poly(A) tail (which means, the last base of the 3' UTR, right?)
I wanted to ask how valid this rule is. I checked and there are more than 2,000 bp between my forward primer and the poly(A) tail. I suppose some of the answers will be "if it works there's probably no problem". But can I trust my results?
I don't know if RT works 100 % (probably not) but RNA gets fragmented and RT enzyme can lose a track. And you only RT fragments from the 3' end with oligod(T), so if you were trying to detect like 5' ends of transcript there would be statistically less of it than 3' ends. So the more far from 3' you have primers the less of the transcript you detect and if your RNA is fragmented a lot that may be serious problem (like if you do FFPE RNA samples). It's hard to say how much this affects the results.
Your friend is right, but reverse polymerase seems pretty good in long haul, so the issue is more with RNA fragmentation. So use random hexamer in RT is always a good idea.