RT-PCR on viral RNA
wlech at medicine.medsch.ucla.edu
Wed Jul 26 13:28:42 EST 1995
One method utilizes generating an RNA copy of your target with a small
(100bp) internal deletion. This is accomplished by designing a
downstream primer (50mer) such that its 3' end binds the plus strand
150bp upstream from the intended target site while its 5' end is
complementary to the original downstream primer binding site.
Amplification, cloning, and in vitro transcription produces an RNA that
is identical to your target except for the 100bp deletion. Use this as
your internal control, using only the one set of original primers which
will amplify both sample RNA and internal control RNA with (presumably)
equal efficiency. (I recommend verifying this "equal" efficiency). The
two bands are easily resolved and, depending on your preferred method of
detection, will provide the necessary quantitation. I am unfamiliar with
the term "high homology standard" in its most strict usage; if the above
is considered such, sorry...
Steven_Burghart at BAYLOR.EDU wrote:
> We are looking for the expression of a small amount of recombinant viral
>RNA's from plant tissue (N. benth). We've read lots of articles, but we don't
>see too many problems with the method that we want to try: we would do a PCR
>with our mutant detection primers and primers for a constant amount of exogenous
>RNA which we would spike into the RT step. The ratio we get on the gel would
>then be "fixed" by a factor accounting for the varying amount of total TMV RNA,
>which we would get just by spectophotometry. In some of the articles we have
>read, the authors have cautioned that two different sets of primers in the same
>reaction can inhibit one another.
> Does anyone see any other problems that we should look out for, or have a
>reliable method for quantifying low levels of viral RNA which seems to produce
>reliable results? Also, has anyone found a way to get around any possible
>primer inhibition, besides using high-homology standards?
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