RT-PCR

Reid, Thomas M. tar9 at NIOBBS1.EM.CDC.GOV
Thu Dec 22 16:20:29 EST 1994


I am attempting to use RT-PCR to amplify the cDNA for the human hprt gene. 
 The source of the RNA is a human lymphoblast cell line. When I use cells 
actively growing in culture I am usually successful.  In fact as few as 100 
cells works fine.  The problem occurs when trying to amplify using frozen 
cells.  The cells ( 10^6 to 10^7) are frozen in a volume of 0.5 ml 
containing tissue culture medium and DMSO.  I have tried using 
phenol-chloroform extraction as well as commercial phenol-guanidinium 
thiocyanate solutions to prepare total RNA.  I use at least 3 ug of RNA 
 along with a gene specific primer for the RT reaction.  The RT reaction is 
diluted 1:5  and 5 ul used for PCR in a total reaction volume of 50 ul.  PCR 
requires 2 rounds of amplification - the second using nested primers.  In 
the majority of cases I get no PCR product.

I would appreciate any suggestions.



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