Protocol Online logo
Top : New Forum Archives (2009-): : PCR, RT-PCR and Real-Time PCR

Reference gene(s) selection and validaton for qPCR - (Apr/22/2014 )

Selecting a proper reference gene(s) for RT-qPCR is an ongoing struggle.

It's easy to "do it", but it's significantly more difficult to do it right.

 

I will dedicate this topic to general discussion about selecting reference (sometimes called housekeeping) genes, software/application used and so on.

 

A web page first.

 

Massively resourced but rather ugly pages concerning normalization and reference genes selection on Gene-Quantification.info site.

 

http://normalisation.gene-quantification.info/

(often contains fulltexts of papers)

 

Some papers.

 

The need for transparency and good practices in the qPCR literature - Nature Methods 2013

Evidence Based Selection of Housekeeping Genes - PloS ONE 2007

 

and of course

 

MIQE Guidelines

 

Today I came accross a free web tool to select reference genes using algorithms from four currently used applications geNorm, Normfinder, BestKeeper, and the comparative ddCt method.

Since I have used only one of them and this requires to only copy Ct directly from the Excel table (!!) it's like a huge help to refgene selection (I used GeNorm for some time, I wouldn't really call it user friendly..)

RefFinder

 

But since I can't compare with the others I don't have any comments on this would be appreciated.

 

(I may add later some sites/papers/... I found highly relevant, post any sources regarding the topic, if you want, too)

-Trof-

Hey Trof, 

       Thanks for quiet useful information. Could you please add material you mentioned in last post?

-Inbox-

I hope I will, but currently I don't have time cause I'm focused on different topic.

 

But if you go through the Gene-Quantification.info site, it contains many many many publications, and was that my starting point too.

-Trof-