title: RefGenes: identification of reliable and condition specific reference genes for RT-qPCR data normalization.
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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Biology, ETH Zurich, 8092 Zurich, Switzerland.
authors.forename: Tomas
authors.initials: T
authors.lastname: Hruz
authors.forename: Markus
authors.initials: M
authors.lastname: Wyss
authors.forename: Mylene
authors.initials: M
authors.lastname: Docquier
authors.forename: Michael W
authors.initials: MW
authors.lastname: Pfaffl
authors.forename: Sabine
authors.initials: S
authors.lastname: Masanetz
authors.forename: Lorenzo
authors.initials: L
authors.lastname: Borghi
authors.forename: Phebe
authors.initials: P
authors.lastname: Verbrugghe
authors.forename: Luba
authors.initials: L
authors.lastname: Kalaydjieva
authors.forename: Stefan
authors.initials: S
authors.lastname: Bleuler
authors.forename: Oliver
authors.initials: O
authors.lastname: Laule
authors.forename: Patrick
authors.initials: P
authors.lastname: Descombes
authors.forename: Wilhelm
authors.initials: W
authors.lastname: Gruissem
authors.forename: Philip
authors.initials: P
authors.lastname: Zimmermann
mesh_terms: D000465:Algorithms; D000818:Animals; D017360:Arabidopsis / Q000235:genetics; D002417:Cattle; D019295:Computational Biology / Q000379:methods; D030541:Databases, Genetic; D005260:Female; D020869:Gene Expression Profiling / Q000379:methods* / Q000592:standards; D006801:Humans; D051379:Mice; D020411:Oligonucleotide Array Sequence Analysis; D012015:Reference Standards; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods / Q000592:standards*; D012984:Software; D013552:Swine; D014584:User-Computer Interface