Competitive/quantitative PCR: radiolabeling of products

Howard R Katz hrk at world.std.com
Fri Oct 22 11:21:06 EST 1993


In reading about competitive PCR to quantify steady-state transcript
levels I note that to obtain radioactive products, some investigators
end-label a primer before the PCR and others instead add a radioactive
dNTP to the PCR reaction with unlabeled primers.  Are there any
advantages/pitfalls to using one method vs. the other in terms of more
accurate quantification of the products?  It seems to me that in each
case, the efficiency of incorporation of the radiolabled primer or dNTP,
respectively, would be the same for the cDNA derived in the RT step from
the competing RNA construct as it is for the cDNA derived from the RNA in
the experimental sample (assuming one adheres to the requirements for
designing an appropriate competitor).  But are there other issues that
might make one labeling method preferable over the other?

Thanks,
Howard



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