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 site.

(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..)



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)


Hey Trof, 

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


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


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


I could add some more to the list


General Help and Quantification Strategies

Critical Factors for Successful Real-Time PCR


Housekeeping Gene Selection

Guideline to reference gene selection for quantitative real-time PCR
Aleksandar Radonic,a Stefanie Thulke, Ian M. Mackay, Olfert Landt,
Wolfgang Siegert, and Andreas Nitschea

Biochemical and Biophysical Research Communications 313 (2004) 856–862.


This paper uses real-time PCR to assay 2-fold gene expression changes (TaqMan-based qRT-PCR)

Real-time quantitative PCR analysis can be used as a primary screen to identify patients with CML treated with imatinib who have BCR-ABL kinase domain mutations.

Susan Branford, Zbigniew Rudzki, Ian Parkinson, Andrew Grigg, Kerry Taylor, John F. Seymour, Simon Durrant, Peter Browett, Anthony P. Schwarer, Chris Arthur, John Catalano, Michael F. Leahy, Robin Filshie, Kenneth Bradstock, Richard Herrmann, David Joske, Kevin Lynch, and Tim Hughes


Real-time PCR for mRNA quantitation
Marisa L. Wong and Juan F. Medrano
BioTechniques Vol. 39, No. 1: pp 75-85 (July 2005)


Impact of RNA quality on reference gene expression stability
Claudina Angela Pérez-Novo, Cindy Claeys, Frank Speleman, Paul Van Cauwenberge, Claus Bachert, and Jo Vandesompele
BioTechniques Vol. 39, No. 1: pp 52-56 (July 2005)


An attempt to produce a standardised rt-qpcr protocol has ben
published in Nature Protocols and can be downloaded from this URL ... 06.236.pdf

-Mad Researcher-

Hi, most reference genes selection I see emphasize on their stable expression across samples. I have a doubt here. Say for example I am to use 3 reference genes in delta delta ct method. In the same sample ( sample a for example) the expression of reference gene 1 is ct 13, reference gene 2 is ct 12, but the ct for reference gene 3 is 8, which is very much different from reference gene 1 and 2. If I am to normalize using all 3 reference gene, would that affect my gene expression assay? I appreciate the help.


Sean Taylor’s Guideline for qPCR