Reverse transcriptase for RT-PCR

Jim Owens 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.
Namely 
>when I do Rt-PCR to amplify RNA and there is some DNA contaminating my
RNA 
>preparation I amplify such DNA .I know the amplification product comes
from 
>DNA because I use primers that span an intron and therefore the 
>amplification product is bigger and because I also get the product when
I do 
>not do the reverse transcription part of the procedure which then
excludes 
>that the amplification comes from hnRNAs.In this particular condition
there 
>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?

Good luck,

Jim Owens



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