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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