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Statistical differences resulting from selection of stable reference genes after hypoxia and hypothermia in the neonatal rat brain

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  • Martín Bustelo
  • Martín A Bruno
  • César F Loidl
  • Manuel Rey-Funes
  • Harry W M Steinbusch
  • Antonio W D Gavilanes
  • D L A van den Hove

Abstract

Real-time reverse transcription PCR (qPCR) normalized to an internal reference gene (RG), is a frequently used method for quantifying gene expression changes in neuroscience. Although RG expression is assumed to be constant independent of physiological or experimental conditions, several studies have shown that commonly used RGs are not expressed stably. The use of unstable RGs has a profound effect on the conclusions drawn from studies on gene expression, and almost universally results in spurious estimation of target gene expression. Approaches aimed at selecting and validating RGs often make use of different statistical methods, which may lead to conflicting results. Based on published RG validation studies involving hypoxia the present study evaluates the expression of 5 candidate RGs (Actb, Pgk1, Sdha, Gapdh, Rnu6b) as a function of hypoxia exposure and hypothermic treatment in the neonatal rat cerebral cortex–in order to identify RGs that are stably expressed under these experimental conditions–using several statistical approaches that have been proposed to validate RGs. In doing so, we first analyzed RG ranking stability proposed by several widely used statistical methods and related tools, i.e. the Coefficient of Variation (CV) analysis, GeNorm, NormFinder, BestKeeper, and the ΔCt method. Using the Geometric mean rank, Pgk1 was identified as the most stable gene. Subsequently, we compared RG expression patterns between the various experimental groups. We found that these statistical methods, next to producing different rankings per se, all ranked RGs displaying significant differences in expression levels between groups as the most stable RG. As a consequence, when assessing the impact of RG selection on target gene expression quantification, substantial differences in target gene expression profiles were observed. Altogether, by assessing mRNA expression profiles within the neonatal rat brain cortex in hypoxia and hypothermia as a showcase, this study underlines the importance of further validating RGs for each individual experimental paradigm, considering the limitations of the statistical methods used for this aim.

Suggested Citation

  • Martín Bustelo & Martín A Bruno & César F Loidl & Manuel Rey-Funes & Harry W M Steinbusch & Antonio W D Gavilanes & D L A van den Hove, 2020. "Statistical differences resulting from selection of stable reference genes after hypoxia and hypothermia in the neonatal rat brain," PLOS ONE, Public Library of Science, vol. 15(5), pages 1-12, May.
  • Handle: RePEc:plo:pone00:0233387
    DOI: 10.1371/journal.pone.0233387
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    References listed on IDEAS

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    1. Venkat Krishnan Sundaram & Nirmal Kumar Sampathkumar & Charbel Massaad & Julien Grenier, 2019. "Optimal use of statistical methods to validate reference gene stability in longitudinal studies," PLOS ONE, Public Library of Science, vol. 14(7), pages 1-18, July.
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    3. Yanqiong Zhang & Dongliang Chen & Michael A Smith & Baohong Zhang & Xiaoping Pan, 2012. "Selection of Reliable Reference Genes in Caenorhabditis elegans for Analysis of Nanotoxicity," PLOS ONE, Public Library of Science, vol. 7(3), pages 1-7, March.
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