primer for RT-PCR

Steven Sullivan sullivan at gwis2.circ.gwu.edu
Sun Mar 5 17:20:26 EST 1995


Paul H. Kim (phk at po.cwru.edu) wrote:
: 	Hi.  I hope this is not a naive question, but I am having some
: reservations about using oligo dT for priming RT reaction in RT.  I am 
: getting good results with it, but it seems to me that random hexamers are 
: prominent.  Is there a reason for this?  Since I am looking for only 
: mRNAs, I thought it would make sense to use oligo dT.  So, are my results
: still valid with oligo dT, or am I limiting my results with this primer?
: Thanks.

Some people advocate random hexamers over oligodT because the latter are
more likely to produce sequences in the 3' untranslated region, whereas
most people want coding region sequence.  But I believe that if you use a
good 'fulllength' reverse transcriptase (e.g. Gibco's Superscript II) then
you'll be able to get well into the coding regions of most mRNAs  (and, 
as you've said, will select against priming off of non-mRNA.)


Steve Sullivan
Anatomy Dept.



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