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Selection of suitable endogenous reference genes for qPCR in kidney and hypothalamus of rats under testosterone influence

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  • Khadijeh Gholami
  • Su Yi Loh
  • Naguib Salleh
  • Sau Kuen Lam
  • See Ziau Hoe

Abstract

Real-time quantitative PCR (qPCR) is the most reliable and accurate technique for analyses of gene expression. Endogenous reference genes are being used to normalize qPCR data even though their expression may vary under different conditions and in different tissues. Nonetheless, verification of expression of reference genes in selected studied tissue is essential in order to accurately assess the level of expression of target genes of interest. Therefore, in this study, we attempted to examine six commonly used reference genes in order to identify the gene being expressed most constantly under the influence of testosterone in the kidneys and hypothalamus. The reference genes include glyceraldehyde-3-phosphate dehydrogenase (GAPDH), actin beta (ACTB), beta-2 microglobulin (B2m), hypoxanthine phosphoribosyltransferase 1 (HPRT), peptidylprolylisomerase A (Ppia) and hydroxymethylbilane synthase (Hmbs). The cycle threshold (Ct) value for each gene was determined and data obtained were analyzed using the software programs NormFinder, geNorm, BestKeeper, and rank aggregation. Results showed that Hmbs and Ppia genes were the most stably expressed in the hypothalamus. Meanwhile, in kidneys, Hmbs and GAPDH appeared to be the most constant genes. In conclusion, variations in expression levels of reference genes occur in kidneys and hypothalamus under similar conditions; thus, it is important to verify reference gene levels in these tissues prior to commencing any studies.

Suggested Citation

  • Khadijeh Gholami & Su Yi Loh & Naguib Salleh & Sau Kuen Lam & See Ziau Hoe, 2017. "Selection of suitable endogenous reference genes for qPCR in kidney and hypothalamus of rats under testosterone influence," PLOS ONE, Public Library of Science, vol. 12(6), pages 1-15, June.
    • Handle: RePEc:plo:pone00:0176368
    DOI: 10.1371/journal.pone.0176368
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    Cited by:

    1. Sanjeeva Herath & Hongying Dai & Jonathan Erlich & Amy YM Au & Kylie Taylor & Lena Succar & Zoltán H Endre, 2020. "Selection and validation of reference genes for normalisation of gene expression in ischaemic and toxicological studies in kidney disease," PLOS ONE, Public Library of Science, vol. 15(5), pages 1-27, May.

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