In article <543v1a$h4a at dfw-ixnews10.ix.netcom.com>,
pjb4 at ix.netcom.com(Peter J Bartholomew) wrote:
> As a minor part of my research project, I isolated total RNA from
> estradiol-treated and control cells followed by RT-PCR. This was done
> to compare the mRNA levels of a certain gene. Since the resultant
> bands seemed of equal intensity following agarose gel electrophoresis
> and ethidium bromide staining, I stated that the estradiol treatment
> did not affect the mRNA levels. In retrospect, I know I should have
> done a northern, but was this conclusion still acceptable?
we're doing a very similar sort of study right now but with about 25-30
different genes. the key to believable semi-quantitative RT-PCR is that
you MUST make sure that your number of amplification cycles is still in
the linear range. if you did 35 cycles or so, my guess is that you were
out of the linear range and couldn't identify any differences that may
have existed (this happens to us all the time).
the way around this is to repeat the PCR and take aliquots of your
reactions at various points in the reaction (i.e. 20, 25 and 30 cycles).
Unless you're using an integrating gel imaging system (e.g. Eagle Eye) you
should also include tracer amounts of a labeled dNTP (we use 32-P a-dCTP)
to make quantitation more accurate (via phosphorimager, densitometry,
finally, i would suggest trying to include primers for a housekeeping gene
(we use GAPDH because we've seen variable actin expression in our system)
in the same reaction as your gene of interest. this will require some
optimization but it makes quantitation much easier since you
(theoretically) have an internal standard. alternatively, you could make
a construct of your gene of interest with a deletion between your RT-PCR
primer sites and use a known amount of that as a competitive target. this
is probably a more accurate internal standard than what we do, but since
we're working with 30+ genes, the construct making is a bit time
consuming. (see Biotechniques 21:268-279 and 280-285(Aug. 1996)) for more
info. on this.
of course, the best way is to just do a northern and be done with it.
Eric C. Anderson
Memorial Sloan-Kettering Cancer Center
1275 York Ave. Box 470
New York, NY 10028
e-anderson at ski.mskcc.org