RT reagents affect PCR

[Michael R. Frank]

shopkins at fs1.ho.man.ac.uk (Steve Hopkins) wrote:
>
> While developing a quantitative PCR method we have become aware that transfer
> of small amounts of the cocktail of reagents in the reverse transcriptase
> reaction (many of which which are present at much higher concentration than
> those in the PCR-mix) influences the subsequent PCR. This means that the PCR
> signal may be a function of the amount of RT-mix transferred, as much as the
> amount of cDNA. It could also be particularly important where cDNA standards, 
> not in RT-mix cocktail, are used. Has anyone experience of what RT-mix factors 
> might be important? Can transfer of random primers affect PCR? We are checking 
> some of these things out, but any help would be appreciated.
> 

We routinely do one-tube rt-pcr, just using a standard pcr buffer, so 
carryover isn't a problem. The conditions might not be optimal for the 
amv rt we use, but it seems to work quite reliably. If you are doing
random priming of the reverse transcription, though, I can see why you
would need to seperate the steps...We generally use a primer which is 
used in the pcr stage anyway. 

Michael Frank
Dept. Plant Biology
University of Illinois, Urbana-Champaign

BTW> I'm looking into doing some quantitative PCR. I would appreciate 
a copy of your procedure if possible. Also open to suggestions from 
anyone with experience in q-PCR. Just email me at mr-frank at uiuc.edu
Thnx!