RT-PCR

[enof]

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,
> Rebecca
> 

QIAGEN has a total RNA kit you can use to quickly prepare total RNA called
RNeasy.


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