RT-PCR

[Shahram Mori]

Reid, Thomas M. (tar9 at NIOBBS1.EM.CDC.GOV) 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 am gonna suggest here that it is not the PCR portion that is giving you
problems. I think it is the RT reaction. Remember that DMSO at amounts
equalling to 17% V/V inhibits RT. Formamide at 5% V/V. 30mMGuanidine-HCL,
 30mM GITC and 1mM EDTA. 
For more detail look up Focus 16 (3): 89, (4): 102.
Cheers,


--
Shahram Mori					   _/\_
Program in Molecular Biology			  _\  /_ Saskatoon/SK/CANADA
Dept. of chemistry and Biochemistry Box 3C	  \_  _/
NMSU  Las Cruces NM				    ||
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