primer for RT-PCR

Shahram Mori smori at nmsu.edu
Mon Mar 6 01:20:20 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.

Depends what you are doing? If you are trying to look at change is a
transcript level that is not polyadenylated ( like histones) then you'll be
missing them completely. There are other transcripts out there that are not
polyadenylated. If you are doing DDRT then I would recommend using random
hexamers because using oligo dT's will result in misalignment which is thought
to be the cause of shadowing and false positives during DDRT. Biotechques
Vol17(2) page226 1994.
I would recommend using random hexamers for RT portion of your rxns.
Cheers
 --
Shahram Mori					   _/\_
Program in Molecular Biology			  _\  /_
Dept. of chemistry and Biochemistry Box 3C	  \_  _/
NMSU  Las Cruces NM				    ||
88003





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