RT-PCR primers and the poly(A) tail
Posted 04 January 2012 - 01:23 PM
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?
Posted 05 January 2012 - 02:00 PM
I never trust anything that can't be doubted.
Posted 06 January 2012 - 07:59 AM