housekeeping gene rtpcr

[Peter Doris]

People interested in this topic might want to see two recent papers 
from our lab on quantitative RT-PCR. This work shows that accurate 
quantitation can be accomplished which meets the mathematical ideals 
mentioned earlier in this thread (Todd Miller). Sam is correct in 
suggesting that it's a lot of work to set up. But once the system is 
validated, you can easily quantitate in single reactions. A critical 
issue in competitive RT-PCR is competitor design and reaction 
product analysis. The issues raised by heteroduplex formation have 
not been well understood, though that is now changing through the 
use of appropriate reaction product analysis techniques.


A. Hayward-Lester, P. J. Oefner and P.A. Doris. Rapid quantification 
of gene expression by competitive RT-PCR and ion-pair reversed-phase 
HPLC. BioTechniques. 20:250-257, 1996.

A. Hayward-Lester, S. Sabatini, P.J. Oefner and P. A. Doris. 
Accurate and absolute quantitative measurement of gene expression by 
single tube RT-PCR and HPLC. Genome Research 5:494-499, 1995.

Peter Doris

Sam Gray wrote:
> 
> jms93 wrote:
> >
> > In article <4eo69l$69d at news.bu.edu>, anttro at bu.edu says...
> > >
> > >We are doing RTPCR using GADPH as a control, and were are finding that
> > even
> > >with equal amounts of rna I am getting varying pcr products of gadph.
> > >Any suggestions? Are housekeeping genes constantly expressed even after
> > >stimulation of cell
> 
> Dear Jon,
> I think the problem that jms93 has with GADPH RTPCR is not a question of
> amplifying the correct sequence but rather one of quantitating mRNA
> levels using GADPH as a constitutively expressed housekeeping control
> gene. I have used B-Actin as an internal control for quantitative RTPCR
> (QRTPCR) and have also found that the expression of B-Actin is not as
> constant as I first thought it would be. This seems to be a problem for
> almost all so-called constitutive housekeeping genes, especially in
> stimulated or developing cell populations (we were interested in murine
> ovaries during development). The only solution is to use an artificially
> constructed internal control RNA such as a deletion mutant of the target
> sequence, cloned into a vector with an RNA polymerase site and generating
> a quantifiable RNA standard. There are several different ways you can go
> about this and unfortunately they are all a bit laborious but if you want
> cast-iron absolute mRNA quanitation by RTPCR this seems to be the only
> way to be sure.
> 
>                 Sam.