Does RNasin inhibit RT?
T. S. Grewal
sasgrewl at reading.ac.uk
Sun Sep 3 08:12:05 EST 1995
I have also tried Promega's RNasin with MMLV (ClonTech) for generating
cDNA to be used in differential display PCR and it did not work.
I have also tried cDNA synthesis without RNase inhibitor and the DDRT-PCR
Recently i tried cDNA synthesis with an RNase inhibitor and MMLV, both
from ClonTech (1st Strand Synthesis Kit), and DDRT-PCT. It worked well
and i got more larger PCR products.
So it seems that if your RNA is of a high quality, you can leave
out the RNase inhibitor, but you do get better results if it is
included. It would seem that certain RNase inhibitors work well
with some reverse transcriptases. Best thing is to find a combination
of inhibtor and enzyme that work well together and stick to it.
(PS. it is possible that Promega's RNasin works well with their reverse
(These are only my views)
On 31 Aug 1995 mhughes at mbcf.stjude.org wrote:
> In article <42079o$kus at cronkite.seas.gwu.edu>, sullivan at gwis2.circ.gwu.edu (Steven Sullivan) writes:
> > In the course of tweaking differential display protocols, it appears that
> > a commonly used RNase inhibitor (Promega RNasin), added as 1 ul per 20 ul
> > reverse transcription reaction, seems to reduce the amount of 1st strand
> > product compared to identical reactions lacking Rnasin. I've found this
> > to be true regardless of whether Superscript or MMLV is used as a reverse
> > transcriptase. Has anyone else observed this, or should I send it off
> > immediately to Biotechniques ? ;>
> We have been using RNasin (Promega) in out RT reactions here for quite
> awhile. We use AMV RT from Life Technologies and don't have a problem getting a
> good deal of cDNA, leaving it out though, does seriously affect our product.
> That might reflect the RNA source more than the reaction. Who would know.
> MHUGHES at mbcf.stjude.org
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