RT-PCR

[Rebecca Schall]

  tar9 at NIOBBS1.EM.CDC.GOV ("Reid, Thomas M.") wrote:

: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.

I have never had much success with isolating RNA from frozen 
cultures.  The process just seems to result in degradation, but there 
is an easy solution.  Just prepare your GITC lysate with your nice 
healthy cells prior to freezing, then store the lysates at -70 degrees C 
until you need them (most protocols say up to a month, I have used 
up to six months with little degradation, and these will certainly work 
for RT-PCR).
Good luck,
Rebecca

Rebecca Schall, Ph.D.
PanVera Corp.
Madison, WI
rebeccas at panvera.com