title: Easy-to-use strategy for reference gene selection in quantitative real-time PCR experiments.
abstract: Real-time PCR is an indispensable technique for mRNA expression analysis but conclusions depend on appropriate reference gene selection. However, while reference gene selection has been a topic of publications, this issue is often disregarded when measuring target mRNA expression. Therefore, we (1) evaluated the frequency of appropriate reference gene selection, (2) suggest an easy-to-use tool for least variability reference gene selection, (3) demonstrate application of this tool, and (4) show effects on target gene expression profiles. All 2015 published articles in Naunyn-Schmiedeberg's Archives of Pharmacology were screened for the use of quantitative real-time PCR analysis and selection of reference genes. Target gene expression (Vegfa, Grk2, Sirt4, and Timp3) in H9c2 cells was analyzed following various interventions (hypoxia, hyperglycemia, and/or isoflurane exposure with and without subsequent hypoxia) in relation to putative reference genes (Actb, Gapdh, B2m, Sdha, and Rplp1) using the least variability method vs. an arbitrarily selected but established reference gene. In the vast majority (18 of 21) of papers, no information was provided regarding selection of an appropriate reference gene. In only 1 of 21 papers, a method of appropriate reference gene selection was described and in 2 papers reference gene selection remains unclear. The method of reference gene selection had major impact on interpretation of target gene expression. With hypoxia, for instance, the least variability gene was Rplp1 and target gene expression (Vefga) heavily showed a 2-fold up-regulation (p = 0.022) but no change (p = 0.3) when arbitrarily using Gapdh. Frequency of appropriate reference gene selection in this journal is low, and we propose our strategy for reference gene selection as an easy tool for proper target gene expression.
authors.affiliation: Klinik für Anästhesiologie & Intensivmedizin, Universität Duisburg-Essen and Universitätsklinikum Essen, Hufelandstr. 55, D-45122, Essen, Germany. Stefanie.Klenke@uk-essen.de.
authors.forename: Stefanie
authors.identifier: http://orcid.org/0000-0002-2500-2809
authors.initials: S
authors.lastname: Klenke
authors.affiliation: Klinik für Anästhesiologie & Intensivmedizin, Universität Duisburg-Essen and Universitätsklinikum Essen, Hufelandstr. 55, D-45122, Essen, Germany.
authors.forename: Kristina
authors.initials: K
authors.lastname: Renckhoff
authors.affiliation: Klinik für Anästhesiologie & Intensivmedizin, Universität Duisburg-Essen and Universitätsklinikum Essen, Hufelandstr. 55, D-45122, Essen, Germany.
authors.forename: Andrea
authors.initials: A
authors.lastname: Engler
authors.affiliation: Klinik für Anästhesiologie & Intensivmedizin, Universität Duisburg-Essen and Universitätsklinikum Essen, Hufelandstr. 55, D-45122, Essen, Germany.
authors.forename: Jürgen
authors.initials: J
authors.lastname: Peters
authors.affiliation: Klinik für Anästhesiologie & Intensivmedizin, Universität Duisburg-Essen and Universitätsklinikum Essen, Hufelandstr. 55, D-45122, Essen, Germany.
authors.forename: Ulrich H
authors.initials: UH
authors.lastname: Frey
mesh_terms: D000818:Animals; D002138:Calibration; D015687:Cell Hypoxia; D002460:Cell Line; D020869:Gene Expression Profiling / Q000592:standards*; D005786:Gene Expression Regulation; D005819:Genetic Markers; D005947:Glucose / Q000378:metabolism; D007530:Isoflurane / Q000494:pharmacology; D032383:Myocytes, Cardiac / Q000187:drug effects / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D060888:Real-Time Polymerase Chain Reaction / Q000592:standards*; D012015:Reference Standards; D015203:Reproducibility of Results
chemical_list: D005819:Genetic Markers; D012333:RNA, Messenger; D007530:Isoflurane; D005947:Glucose
keywords: H9c2 cells; Hypoxia; Isoflurane, preconditioning; Quantitative real-time PCR; Reference gene