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

Springer Science and Business Media LLC - Tập 12 Số 1 - 2011
Tomás Hrúz1, Markus Wyss1, Mylène Docquier2, Michael W. Pfaffl3, Sabine Masanetz3, Loris Borghi4, Phebe Verbrugghe5, Luba Kalaydjieva5, Stefan Bleuler6, Oliver Laule6, Patrick Descombes2, Wilhelm Gruissem4, Philip Zimmermann4
1Institute of Theoretical Computer Science, ETH Zurich, 8092 Zurich, Switzerland
2Genomics Platform, NCCR 'Frontiers in Genetics', University of Geneva, 1211, Geneva 4, Switzerland
3Physiology Weihenstephan, TUM, Weihenstephaner Berg 3, 85354, Freising, Germany
4Department of Biology, ETH Zurich, 8092, Zurich, Switzerland
5Western Australian Institute for Medical Research, UWA Centre for Medical Research, Perth, Australia
6NEBION AG, Hohlstrasse 515, 8048, Zurich, Switzerland

Tóm tắt

Abstract Background 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. Results 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. Conclusion 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 http://www.genevestigator.com.

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