Reverse transcriptase for RT-PCR
jow at helix.nih.gov
Fri Jul 9 08:12:33 EST 1993
In article <1993Jul8.142736.20269 at galileo.cc.rochester.edu> RICHARD P.
PHIPPS, PHIP at BPHVAX.BIOPHYSICS.ROCHESTER.EDU writes:
> I've got quite a bit of experience with
> RT-PCR using USB MMLV-RT, and I was wondering if anyone is using
> RNAse H(-) RTs such as superscript from BRL.
> Is it worth the extra expense for this type of assay?
I have no personal experience using RNaseH(-) RTase, but, when I was
reading about RT-PCR before trying it, there was someone who wrote that
the RNaseH activity was helpful since RNA could inhibit TaqPolymerase.
Here are some comments from a few days back:
>Biotechniques 14(1), 24-25 (1993) has an article describing suppression
>of PCR by *high* levels of RNA
>I do not think that RNA can interfere with DNA amplification. I did not
>address that question specifically but I have some indirect evidence.
>when I do Rt-PCR to amplify RNA and there is some DNA contaminating my
>preparation I amplify such DNA .I know the amplification product comes
>DNA because I use primers that span an intron and therefore the
>amplification product is bigger and because I also get the product when
>not do the reverse transcription part of the procedure which then
>that the amplification comes from hnRNAs.In this particular condition
>is "a lot of RNA" and probably few DNA templates and nevertheless I can
>amplify them (unfortunately!!). All this in mamalian cells extracts...I
>think for the purpose of the question that it is not important.
Of course, the second comment says nothing about the RNaseH activity of
the RTase used!
Also, a few weeks ago there was someone using Superscript RTase who was
having problems with their RT-PCR. That may not have been the problem,
since that person did not heat-kill the RTase, and RTase does inhibit
TaqPolymerase (Sellner et al. Nucl Acids Res 20:1487(1992)).
So the extra expense is probably not worth it. If your system is
working nicely, why mess with success?
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