In article... pjb4 at ix.netcom.com(Peter J Bartholomew) wrote:
>... 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?
For a number of reasons, RT-PCR will fail to detect true differences in
the level of expression of an mRNA; I'll explain two of these reasons. The
first may be that genomic (DNA) contamination of your RNA sample may
provide template for PCR in your control samples. To avoid this, DNAse
treatment of the RNA prior to cDNA synthesis (with RQ1 RNAse-free DNAse)
is recommended. Also, you should include a PCR negative control using as
template the RNA that has not been reverse transcribed. Any band that
appears in this sample lane must be of contaminating origin (ie genomic).
The next reason is that standard (ie non-competitive PCR) results in the
accumulation of amplified product that does not follow the predicted
exponential curve. One explanation of this is the Cot effect. Stated
simply, as [product] accumulates and [primers] diminishes, the strands of
DNA begin to preferentially re-anneal instead of anneling with the
primers. This occurs earlier with messages that were present in higher
concentration at the start of the reaction, so it tends to minimize the
molar ratio difference between amplified products and starting templates.
The effect is accentuated with high number of cycles.
Yes, for quantitation and comparisons, Northern is better. However, given
its low sensitivity for rare messages, ribonuclease protection assay or
competitive PCR may be neccessary if the message you are looking for is
rare. Standard PCR is ok for showing true on/off changes in gene
expression, but not for up- or down-regulation of a gene. The methods of
quantifying the intensity of the band will also affect your accuracy. For
eg, agarose gel and the naked eye in your case, is obviously less accurate
than hot pcr and phosphorimaging the product.
Louis Alarcon, M.D.
University of Pittsburgh
Department of Surgery