Primers NOT needed for RT rx'n?!?!
hamel at cc.umanitoba.ca
Sat Oct 29 20:14:22 EST 1994
There is an intriguing article in the recent issue of Nucleic Acids
Research, that I wish to solicit responses towards (PLEASE post, if not,
I'll summarize all email that I receive on this matter).
The article in question is;
Barbara Frech & Ernst Peterhans. 1994. "RT-PCCR: 'background priming'
during reverse transcription. NAR. Vol.22, No.20, pg 4324-4343.
Anyone (else) out there care to "take a stab at this one"?
Jest of their paper is that total cellular RNA ALONE is sufficient for
priming of cDNA synthesis by reverse transcripase ... diverse cDNAs ...
thus, on surface all one needs do is "chick in" a bunch of total RNA (they
use between 1 ug and 10 ug total cellular RNA) in 50 uL RT rx'n (MMLV or
AMV RTs work okay), incubate as usual (up to 42*C for up to 1.5 hrs), then
use aliquot of this for PCR involving sequence specific primer.
Total cellular RNA from VERY different (divergent) species will work ...
for example, even yeast total RNA (but NOT yeast tRNA) will act as "primer"
for driving RT rx'n of mammalian RNA. ... whereas random hexamers don't
work NEARLY as well ... sequence specific primers work SLIGHTLY better
than does total RNA "primers" ...
Their results are intriguing to say the least (perhaps unsettling,
unerving, etc? :^) ... or, with sound mind (or maybe wild imagination is
needed instead?), not too surprising, afterall? 8^)
First question about this particular publication that came to my mindd is
whether they sequenced any of these RT-PCR products? Second question was
whether same degree of thoroughness used for studying BVD virus RT-PCR was
also undertaken for studying GAPDH RT-PCR (they are from a vet. virology
lab, as am I, thus, BVDV is obviously main interest) ... I also study BVDV
(we now use RT-PCR for routine diagnosis), & will thus CERTAINLY try to
replicate their study (using 3 completely different sets of primers ... one
set from 5' UTR, second set from middle, third set from near 3' end, of
BVDV genome ... will also perform same study on other RT-PCRs ... I will
thus be studying a total of about 20 different RT-PCRs using their "total
cellular RNA as primer" for RT rx'n, followed by sequence specific primer
PCR. ... we currently use one tube RT-PCR, wherein RT & Taq enzymes & both
primers are in same rx'n mix & for past 2 years now, all of our RNAs have
been extracted with yeast total RNA as carrier since we've found that yeast
total RNA not only improved RNA yield but also improves yield of RT-PCR
product ... who would have thought of trying RT rx'n in ABSENCE of sequence
specific primers? ... I certainly didn't!! :^)
**** I would also appreciate if someone could help me contact the authors
by email (assuming they use email ... my searches for their email addresses
using various internet tools came up empty ... 2 hours gophering at best of
my ability & experience, netfind, etc).
On surface this article would seem to bode very well for various
strategies used for increasing specificity of cDNA priming in RT-PCR ...
for example, using immobilized template RNA (bound onto membrane
or beads, instead of usually being freely in solution).
How many out there care to share their experiences & opinions in using
immobilized template RNA for RT rx'ns & whether you see significant
improvements in spcificity of RT rx'n (and therefore, also of RT-PCR)?
(ps, many thanks to my good friend Roland Hubner for bringing this
article to my attention, literally (he faxed a copy to me from Belgium :^)
Andre Hamel email: hamel at cc.umanitoba.ca
Manitoba Gov't Dept of Agriculture tel: 204/945-7630
Veterinary Services Branch FAX: 204/945-8062
Winnipeg, Manitoba, CANADA
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