RT-PCR on viral RNA

Steven_Burghart at BAYLOR.EDU Steven_Burghart at BAYLOR.EDU
Mon Jul 24 09:17:13 EST 1995

     We are looking for the expression of a small amount of recombinant viral 
RNA's from plant tissue (N. benth).  We've read lots of articles, but we don't 
see too many problems with the method that we want to try:  we would do a PCR 
with our mutant detection primers and primers for a constant amount of exogenous 
RNA which we would spike into the RT step.  The ratio we get on the gel would 
then be "fixed" by a factor accounting for the varying amount of total TMV RNA, 
which we would get just by spectophotometry.  In some of the articles we have 
read, the authors have cautioned that two different sets of primers in the same 
reaction can inhibit one another.  
     Does anyone see any other problems that we should look out for, or have a 
reliable method for quantifying low levels of viral RNA which seems to produce 
reliable results?  Also, has anyone found a way to get around any possible 
primer inhibition, besides using high-homology standards?

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