Quantitative PCR

Todd Miller - Pharmacology tmiller at newssun.med.miami.edu
Tue Aug 29 13:59:16 EST 1995


In article <thomas.kreuzer.62.0012F0A2 at kfunigraz.ac.at>,
Thomas Kreuzer <thomas.kreuzer at kfunigraz.ac.at> wrote:
>On Thu. 24 Aug Frank Chen wrote as a reply to a question concerning 
>quantitative RT-PCR:
>
>" No matter how you do it and how good your controls are , your results from 
>quantitative PCR will be questionable."
>
>Since I am trying to put up a quantitative RT-PCR - method myself (and being 
>quite new to the method of PCR in common) I would be interested in the major 
>reservations against the principle of quantitative PCR and especially 
>quantitative RT-PCR. What exactly makes it´s results questionable and why 
>can´t these problems be avoided ?
>It would be a great help if you could also give me some advice about 
>literature dealing with these problems.
>
I would highly recommend reading an article by Luc Raeymaekers in
_Analytical Biochemistry_ (I think it is volume 214, 1993, but you
may have to check your favorite citation source).  He goes through
all the mathematical theory of quantitative competitive PCR and comes
to the conclusion that there are many papers published that have
ignored some basic theory and predictions, which makes the data
very suspicious.  Basically, the assumption that your competitor and
your target have equal amplification efficiencies is almost never
borne out, and this results in huge errors by the time you go through
all the cycles.  

Quantitative PCR works only for HIV and AIDS patients...  ;-)

Todd Miller, PhD
University of Miami School of Medicine
tmiller at newssun.med.miami.edu

 
>Thanks in advance,
>
>Tom Kreuzer





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