RT-problem in RT-PCR

Bill Alexander alexanderw at cber.cber.fda.gov
Thu Feb 22 15:41:08 EST 1996


In Article <312D1692.3E3C at kfunigraz.ac.at>,
thomas.kreuzer at BALU.KFUNIGRAZ.AC.AT (Thomas Kreuzer) wrote:
>Hi, all you methods and reagents, 
>
>I have already posted this under a more specific title,but so far I have 
>received no responses. But I am sure that there is someone in this newsgroup 
>who can help me with my little problem.
>I am STILL looking for literature concerning the efficiency of reverse 
>transcriptase when transcribing different mRNA targets.
>I have found a paper by Berger et al. telling me that the reverse 
>transcriptase works with an efficiency of 50 %  under optimal conditions i.e. 
>only 50 % of input mRNA are transcribed into full-lenght cDNA , which answers 
>question a) in my last posting  = ( How many cDNA copies can be produced for 
>each molecule of mRNA in one hour of reverse transcription? )
>But I did not find anything concerning question b): Can I assume that 
>different mRNAs are transcribed at the same rate when using oligo-p(dT) 
>primers or can there be differences in the transcription rate due to 
>sequential or structural differences ?

I don't know why anyone would assume this.  Try: Ferre, Francois;
"Quantitative or Semi-Quantitative PCR: Reality Versus Myth",  PCR Methods
and Applications, Vol. 2, No. 1, pp. 1-9.   This paper does talk about the
limitations of RT-PCR.  Many people try to make the above assumption and
seem to get their papers published without any problems.  If you really want
to quantitate your gene's expression there are a lot of necessary controls,
if you just want to publish, it seems like you can get away with anything.

There are also papers shooting down the so called reporter genes but I am
very tired of this subject.

>It is quite important for me to get an answer to that question and I am sure 
>that some quantitative-RT-PCR-guru in this newsgroup knows it. So if you have 
>literature references , personal experiences or opinions , please send me an 
>email or post to this newsgroup.
>
>Thanks in advance,
>
>Thomas Kreuzer
>Inst.f. Biochemistry
>University of Graz 
>Austria
Regards,

Bill Alexander
alexanderw at cber.cber.fda.gov

     "640K ought to be enough for anybody." -- Bill Gates, 1981



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