enof at kaiwan.com
Tue Dec 27 17:51:16 EST 1994
In article <5DF30F75D25 at mercury.panvera.com>, REBECCAS at PANVERA.COM
("Rebecca Schall") wrote:
> 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,
QIAGEN has a total RNA kit you can use to quickly prepare total RNA called
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