quantitative PCR

Dr Andrew J. Doherty doherty at bsa.bristol.ac.uk
Fri Jul 19 02:33:23 EST 1996


Martin Offterdinger wrote:
> 
> Hello!
> 
> I am considering setting up a quantitative RT-PCR to measure induction of mRNA of low expressed genes.
> I would like to know, which internal standard for normalisation I should use. I have got a fragment of the cDNA of the RNA I want to measure in pGEM, so I could in vitro transscribe.
> I would also be grateful for any addititonal information about this method, which other controls are important etc...
> 
> Thank you very much in advance
> 
> Martin Offterdinger, E-mail:a8803349 at unet.univie.ac.at
Hi Martin,

Certainly the best kind of internal control for qRT-PCR is a cRNA which
uses the same primers as the mRNA you want to measure. You can either
make it a slightly different size or introduce a unique restriction site
which would cut your control in half. These simply allow you to
distinguish between the internal control and the mRNA you want to
measure.

A second approach would be to use some sort of housekeeping gene to
normalise your mRNA of interest to. Whichever gene you choose, it would
have to be constitutivey expressed. In looking at GABAa subunit
expression, I used ß-Actin. If you can get the kinetics of the two
reactions into line, you can also use the expression of this gene to
take into account variability in your mRNA preparations. However, this
will only give you a measure of the change in mRNA levels and not an
absolute value for the amount of any mRNA in your cells, unlike
competitive RT-PCR. So it really dependes on what you want as an end
result. If I was setting up a qRT-PCR method again, I would almost
certainly go down the competitive route, using a modified cRNA of the
gene of interest - and that's my twopenneth worth

Hope this helps
TTFN

-- 
***********************************************
   Dr Andrew Doherty                          
   Department of Anatomy                   
   School of Medical Sciences             
   University Walk                               
   Bristol                                            
   UK
   BS8 1TD

   e-mail  Doherty at bsa.bristol.ac.uk

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