Semi-quantitative RT-PCR control

June Eva Paciga jpaciga at com1.med.usf.edu
Mon Jul 21 16:57:46 EST 1997


Martin Offterdinger wrote:
> 
> On 9 Jul 1997 04:23:03 -0700, akirby at eastman.ucl.ac.uk (Alun Kirby)
> wrote:
> 
> >Hi.
> >I'm trying to start doing semi-quantitative RT-PCR in human samples and
> >need a control reaction to confirm equivalent starting cDNA levels in
> >each sample.
> >I was going to use beta-actin but have been told that this varies
> >between tissues. Any info on which primers to use and for what template
> >would be gratefully appreciated. GAPDH perhaps?
> >
> >Thanks.....Alun.
> >
> >e-mail to akirby at eastman.ucl.ac.uk
> 
> You could use 36B4, a gene fragment that codes for a ribosomal
> protein; this should  be expressed homogeneously.
> In my opinion creation of a mutated form of your cDNA and competitive
> amplification is still the best way of quantification.
> Martin

Check out Ambion's "QuantumRNA" Quantitative RT-PCR Module. You have a
choice of using two different quantitative techniques, relative or
competitive rt-pcr.  I use the Relative RT-PCR technique, by which you
can compare the relative level of your target PCR product between
multiple samples.  This technique uses 18S rRNA as an internal control.
I am very satisfied with the results I have been getting, I think this
is a great product.  For the detailed explaination and information go
to:
	http://www.ambion.com/RNA/html/quant-w-rtpcr.html

Hope this helps.  And please note, I am not affiliated with Ambion.

June Eva Paciga
University of South Florida
Department Pathology
Tampa FL



More information about the Methods mailing list