title: Selection of reference genes for gene expression studies in rats.
abstract: Selection of the most stable reference gene is critical for a reliable interpretation of gene expression data using RT-PCR. In order so, 17 commonly used genes were analyzed in Wistar rat duodenum, jejunum, ileum and liver following a fat gavage and at two time periods. These reference genes were also tested in liver from Zucker (fa/fa) on a long-term dietary trial. Four strategies were used to select the most suitable reference gene for each tissue: ranking according to biological coefficient of variation and further validation by statistical comparison among groups, geNorm, NormFinder and BestKeeper programs. No agreement was observed among these approaches for a particular gene, nor a common gene for all tissues. Furthermore we demonstrated that normalising using an inadequate reference conveyed into false negative and positive results. The selection of genes provided by BestKeeper resulted in more reliable results than the other statistical packages. According to this program, Tbp, Ubc, Hprt and Rn18s were the best reference genes for duodenum, jejunum, ileum and liver, respectively following a fat gavage in Wistar rats and Rn18s for liver in another rat strain on a long-term dietary intervention. Therefore, BestKeeper is highly recommendable to select the most stable gene to be used as internal standard and the selection of a specific reference expression gene requires a validation for each tissue and experimental design.
authors.affiliation: Departamento de Bioquímica y Biología Molecular y Celular, Facultad de Veterinaria, Instituto de Investigación Sanitaria de Aragón-Universidad de Zaragoza, Spain.
authors.forename: Roberto
authors.initials: R
authors.lastname: Martínez-Beamonte
authors.forename: María A
authors.initials: MA
authors.lastname: Navarro
authors.forename: Ana
authors.initials: A
authors.lastname: Larraga
authors.forename: Mark
authors.initials: M
authors.lastname: Strunk
authors.forename: Cristina
authors.initials: C
authors.lastname: Barranquero
authors.forename: Sergio
authors.initials: S
authors.lastname: Acín
authors.forename: Mario A
authors.initials: MA
authors.lastname: Guzman
authors.forename: Pablo
authors.initials: P
authors.lastname: Iñigo
authors.forename: Jesús
authors.initials: J
authors.lastname: Osada
mesh_terms: D000818:Animals; D004386:Duodenum / Q000378:metabolism; D015870:Gene Expression; D007082:Ileum / Q000378:metabolism; D007583:Jejunum / Q000378:metabolism; D008099:Liver / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D012015:Reference Standards; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000592:standards*; D046888:Tissue Array Analysis / Q000379:methods