Quantitative RT-PCR

Peter-Bram 't Hoen p.hoen at lacdr.leidenuniv.nl
Tue Feb 23 11:49:02 EST 1999


Hi,

A number of excellent reviews have been published recently on the topic of
quantitative RT-PCR.
In: Quantitative RT-PCR: pitfalls and potential. Biotechniques 26: 112-125
(January 1999) by Freeman et al., a lot of theoretical and practical
considerations are summarised which are important to look at before
developing a quantitative RT-PCR assay.
In my research I use RT-PCR very frequently and I can get very reliable
results when I add 32P-labelled alpha dCTP to my PCR reactions which makes
quantification with phosphorimaging software possible. Making the assay
radioactive increases the sensitivity compared to ordinary ethidium bromide
staining of the PCR products and enables to quantify PCR products at low PCR
cycles (10-20) which is necessary to stay in the logarithmic phase of the PCR
reaction.

Good luck,

Peter


asheat at my-dejanews.com wrote:

> Dear Deja members
>
> I want to do quantitative RT-PCR for cytokine, Can any one suggest how to
> start  and can anybody suggest  some reference.
>
> Thanks in advance
>
> -----------== Posted via Deja News, The Discussion Network ==----------
> http://www.dejanews.com/       Search, Read, Discuss, or Start Your Own



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