Expressed repeat elements improve RT-qPCR normalization across a wide range of zebrafish gene expression studies

PLoS One. 2014 Oct 13;9(10):e109091. doi: 10.1371/journal.pone.0109091. eCollection 2014.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Gene Expression
  • Gene Expression Profiling / methods
  • Gene Expression Profiling / standards*
  • Real-Time Polymerase Chain Reaction / methods
  • Real-Time Polymerase Chain Reaction / standards*
  • Reference Standards
  • Zebrafish / genetics*

Grants and funding

This work was supported by Ghent University (Methusalem grant BOF08/01M01108 to A.D.P., GOA-UGent grant no. 12051203 to F.S.), by the Fund for Scientific Research – Flanders (FWO) (grant no. G.0574.13 to P.C.), by the Belgian Program of Interuniversity Poles of Attraction IUAP (to F.S.) and by the “Stichting tegen kanker” (grant no. 365O9110 to F.S.). S.V. is supported by a PhD fellowship from the Fund for Scientific Research – Flanders (FWO). G.V.P. and A.R. are supported by a PhD fellowship from the Ghent University research fund (BOF). P.R. has a postdoctoral research grant from the Fund for Scientific Research – Flanders (FWO). A.W. and S.L. are postdoctoral fellows supported by the Special Research Fund (BOF) of Ghent University. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.