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Mammalian Genome

, Volume 29, Issue 9–10, pp 656–662 | Cite as

Identification of reference genes suitable for RT-qPCR studies of murine gastrulation and patterning

  • Kristen S. Barratt
  • Koula E. M. Diamand
  • Ruth M. Arkell
Article

Abstract

Quantitative reverse transcriptase PCR (RT-qPCR), a powerful and efficient means of rapidly comparing gene expression between experimental conditions, is routinely used as a phenotyping tool in developmental biology. The accurate comparison of gene expression across multiple embryonic stages requires normalisation to reference genes that have stable expression across the time points to be examined. As the embryo and its constituent tissues undergo rapid growth and differentiation during development, reference genes known to be stable across some time points cannot be assumed to be stable across all developmental stages. The immediate post-implantation events of gastrulation and patterning are characterised by a rapid expansion in cell number and increasing specialisation of cells. The optimal reference genes for comparative gene expression studies at these specific stages have not been experimentally identified. In this study, the expression of five commonly used reference genes (H2afz, Ubc, Actb, Tbp and Gapdh) was measured across murine gastrulation and patterning (6.5–9.5 dpc) and analysed with the normalisation tools geNorm, Bestkeeper and Normfinder. The results, validated by RT-qPCR analysis of two genes with well-documented expression patterns across these stages, indicated the best strategy for RT-qPCR studies spanning murine gastrulation and patterning utilises the concurrent reference genes H2afz and Ubc.

Notes

Acknowledgements

This work was supported by a National Health and Medical Research Council Grant [#1126288 to R.M.A.].

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

All procedures involving animals performed in this study were in accordance with The Australian National University Animal Experimentation Ethics Committee standards.

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Early Mammalian Development Laboratory, John Curtin School of Medical ResearchThe Australian National UniversityCanberraAustralia

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