title: Expressed repeat elements improve RT-qPCR normalization across a wide range of zebrafish gene expression studies.
abstract: The selection and validation of stably expressed reference genes is a critical issue for proper RT-qPCR data normalization. In zebrafish expression studies, many commonly used reference genes are not generally applicable given their variability in expression levels under a variety of experimental conditions. Inappropriate use of these reference genes may lead to false interpretation of expression data and unreliable conclusions. In this study, we evaluated a novel normalization method in zebrafish using expressed repetitive elements (ERE) as reference targets, instead of specific protein coding mRNA targets. We assessed and compared the expression stability of a number of EREs to that of commonly used zebrafish reference genes in a diverse set of experimental conditions including a developmental time series, a set of different organs from adult fish and different treatments of zebrafish embryos including morpholino injections and administration of chemicals. Using geNorm and rank aggregation analysis we demonstrated that EREs have a higher overall expression stability compared to the commonly used reference genes. Moreover, we propose a limited set of ERE reference targets (hatn10, dna15ta1 and loopern4), that show stable expression throughout the wide range of experiments in this study, as strong candidates for inclusion as reference targets for qPCR normalization in future zebrafish expression studies. Our applied strategy to find and evaluate candidate expressed repeat elements for RT-qPCR data normalization has high potential to be used also for other species.
authors.affiliation: Center of Medical Genetics, Ghent University, Ghent, Belgium.
authors.forename: Suzanne
authors.initials: S
authors.lastname: Vanhauwaert
authors.affiliation: Center of Medical Genetics, Ghent University, Ghent, Belgium.
authors.forename: Gert
authors.initials: G
authors.lastname: Van Peer
authors.affiliation: Center of Medical Genetics, Ghent University, Ghent, Belgium.
authors.forename: Ali
authors.initials: A
authors.lastname: Rihani
authors.affiliation: Center of Medical Genetics, Ghent University, Ghent, Belgium.
authors.forename: Els
authors.initials: E
authors.lastname: Janssens
authors.affiliation: Center of Medical Genetics, Ghent University, Ghent, Belgium.
authors.forename: Pieter
authors.initials: P
authors.lastname: Rondou
authors.affiliation: Center of Medical Genetics, Ghent University, Ghent, Belgium.
authors.forename: Steve
authors.initials: S
authors.lastname: Lefever
authors.affiliation: Center of Medical Genetics, Ghent University, Ghent, Belgium.
authors.forename: Anne
authors.initials: A
authors.lastname: De Paepe
authors.affiliation: Center of Medical Genetics, Ghent University, Ghent, Belgium.
authors.forename: Paul J
authors.initials: PJ
authors.lastname: Coucke
authors.affiliation: Center of Medical Genetics, Ghent University, Ghent, Belgium.
authors.forename: Frank
authors.initials: F
authors.lastname: Speleman
authors.affiliation: Center of Medical Genetics, Ghent University, Ghent, Belgium.
authors.forename: Jo
authors.initials: J
authors.lastname: Vandesompele
authors.affiliation: Center of Medical Genetics, Ghent University, Ghent, Belgium.
authors.forename: Andy
authors.initials: A
authors.lastname: Willaert
mesh_terms: D000818:Animals; D015870:Gene Expression; D020869:Gene Expression Profiling / Q000379:methods / Q000592:standards*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods / Q000592:standards*; D012015:Reference Standards; D015027:Zebrafish / Q000235:genetics*