Open Access Highly Accessed Open Badges Methodology article

RefGenes: identification of reliable and condition specific reference genes for RT-qPCR data normalization

Tomas Hruz2, Markus Wyss2, Mylene Docquier3, Michael W Pfaffl4, Sabine Masanetz4, Lorenzo Borghi1, Phebe Verbrugghe6, Luba Kalaydjieva6, Stefan Bleuler5, Oliver Laule5, Patrick Descombes3, Wilhelm Gruissem1 and Philip Zimmermann1*

Author Affiliations

1 Department of Biology, ETH Zurich, 8092 Zurich, Switzerland

2 Institute of Theoretical Computer Science, ETH Zurich, 8092 Zurich, Switzerland

3 Genomics Platform, NCCR 'Frontiers in Genetics', University of Geneva, 1211 Geneva 4, Switzerland

4 Physiology Weihenstephan, TUM, Weihenstephaner Berg 3, 85354 Freising, Germany

5 NEBION AG, Hohlstrasse 515, 8048 Zurich, Switzerland

6 Western Australian Institute for Medical Research, UWA Centre for Medical Research, Perth, Australia

For all author emails, please log on.

BMC Genomics 2011, 12:156  doi:10.1186/1471-2164-12-156

Published: 21 March 2011



RT-qPCR is a sensitive and increasingly used method for gene expression quantification. To normalize RT-qPCR measurements between samples, most laboratories use endogenous reference genes as internal controls. There is increasing evidence, however, that the expression of commonly used reference genes can vary significantly in certain contexts.


Using the Genevestigator database of normalized and well-annotated microarray experiments, we describe the expression stability characteristics of the transciptomes of several organisms. The results show that a) no genes are universally stable, b) most commonly used reference genes yield very high transcript abundances as compared to the entire transcriptome, and c) for each biological context a subset of stable genes exists that has smaller variance than commonly used reference genes or genes that were selected for their stability across all conditions.


We therefore propose the normalization of RT-qPCR data using reference genes that are specifically chosen for the conditions under study. RefGenes is a community tool developed for that purpose. Validation RT-qPCR experiments across several organisms showed that the candidates proposed by RefGenes generally outperformed commonly used reference genes. RefGenes is available within Genevestigator at webcite.