Evaluation of the Housekeeping Genes; β‐Actin, Glyceraldehyde‐3‐Phosphate‐Dehydrogenase, and 18S rRNA for Normalization in Real‐Time Polymerase Chain Reaction Analysis of Gene Expression in Human Adipose Tissue
Archives of Medical Laboratory Sciences,
Vol. 4 No. 3 (2018),
28 December 2019
https://doi.org/10.22037/amls.v4i3.26269
Abstract
Background: Several studies suggested that beta-actin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and 18S rRNA are expressed constitutively and contribute to the fundamental reference actions essential for cell viability and maintenance. However, there are inconsistency in this regard. Hence, we aimed to evaluate the accuracy of these three potential reference genes for Real‐Time quantitative reverse transcriptase‐polymerase chain reaction (qRT‐PCR) application for normalization in two types of human adipose tissues.
Materials and Methods: Subcutaneous and visceral fat tissues were derived from 19 healthy and 20 obese subjects and RT-qPCR was applied to determine the expression levels of beta-actin, GAPDH, and18S rRNA.
Results: The gene expression level of beta-actin, GAPDH, and 18S rRNA was essentially the same in the subcutaneous and visceral fat tissues of all participants (P>0.05). Hence, all considered housekeeping genes displayed high expression stability and the analysis revealed that normalization to all of these three housekeeping genes gave a result that satisfactorily reflected the acceptable mRNA expression levels in adipose tissues.
Conclusion: Collectively, our findings suggest of beta-actin, GAPDH, and18S rRNA as reference genes applicable in human adipose tissue in the context of obesity.
- Housekeeping gene
- Beta-actin
- Glyceraldehyde-3-phosphate dehydrogenase (GAPDH)
- 18S rRNA
- Real‐time quantitative reverse transcriptase‐polymerase chain reaction (RT‐qPCR)
How to Cite
References
Hibbeler, S., J.P. Scharsack, and S. Becker, Housekeeping genes for quantitative expression studies in the three-spined stickleback Gasterosteus aculeatus. BMC Molecular Biology, 2008. 9(1): p. 18.
Bustin, S.A., Quantification of mRNA using real-time reverse transcription PCR (RT-PCR): trends and problems. J Mol Endocrinol, 2002. 29(1): p. 23-39.
Iovanna, J.L., et al., Changes in growth and pancreatic mRNA concentrations during postnatal development of rat pancreas. Pancreas, 1990. 5(4): p. 421-6.
Goldsworthy, S.M., et al., Variation in expression of genes used for normalization of Northern blots after induction of cell proliferation. Cell Prolif, 1993. 26(6): p. 511-8.
Hibbeler, S., J.P. Scharsack, and S. Becker, Housekeeping genes for quantitative expression studies in the three-spined stickleback Gasterosteus aculeatus. BMC Molecular Biology, 2008. 9(1): p. 18.
Pfaffl, M.W., et al., Determination of stable housekeeping genes, differentially regulated target genes and sample integrity: BestKeeper – Excel-based tool using pair-wise correlations. Biotechnology Letters, 2004. 26(6): p. 509-515.
Suzuki, T., P.J. Higgins, and D.R. Crawford, Control selection for RNA quantitation. Biotechniques, 2000. 29(2): p. 332-7.
Bustin, S.A., Quantification of mRNA using real-time reverse transcription PCR (RT-PCR): trends and problems. J Mol Endocrinol, 2002. 29(1): p. 23-39.
Matrisian, L.M., et al., Epidermal growth factor and oncogenes induce transcription of the same cellular mRNA in rat fibroblasts. Embo j, 1985. 4(6): p. 1435-40.
Iovanna, J.L., et al., Changes in growth and pancreatic mRNA concentrations during postnatal development of rat pancreas. Pancreas, 1990. 5(4): p. 421-6.
Kanayama, S. and R.A. Liddle, Influence of food deprivation on intestinal cholecystokinin and somatostatin. Gastroenterology, 1991. 100(4): p. 909-15.
Goldsworthy, S.M., et al., Variation in expression of genes used for normalization of Northern blots after induction of cell proliferation. Cell Prolif, 1993. 26(6): p. 511-8.
Foss, D.L., M.J. Baarsch, and M.P. Murtaugh, Regulation of hypoxanthine phosphoribosyltransferase, glyceraldehyde-3-phosphate dehydrogenase and beta-actin mRNA expression in porcine immune cells and tissues. Anim Biotechnol, 1998. 9(1): p. 67-78.
Chechi, K., et al., Validation of Reference Genes for the Relative Quantification of Gene Expression in Human Epicardial Adipose Tissue. PLOS ONE, 2012. 7(4): p. e32265.
Bustin, S.A., Quantification of mRNA using real-time reverse transcription PCR (RT-PCR): trends and problems. J Mol Endocrinol, 2002. 29(1): p. 23-39.
Bustin, S.A., Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays. J Mol Endocrinol, 2000. 25(2): p. 169-93.
Glare, E.M., et al., beta-Actin and GAPDH housekeeping gene expression in asthmatic airways is variable and not suitable for normalising mRNA levels. Thorax, 2002. 57(9): p. 765-70.
Gabrielsson, B.G., et al., Evaluation of reference genes for studies of gene expression in human adipose tissue. Obes Res, 2005. 13(4): p. 649-52.
Perez, L.J., et al., Validation of optimal reference genes for quantitative real time PCR in muscle and adipose tissue for obesity and diabetes research. Sci Rep, 2017. 7(1): p. 3612.
Gorzelniak, K., et al., Validation of endogenous controls for gene expression studies in human adipocytes and preadipocytes. Horm Metab Res, 2001. 33(10): p. 625-7.
Schmittgen, T.D. and B.A. Zakrajsek, Effect of experimental treatment on housekeeping gene expression: validation by real-time, quantitative RT-PCR. J Biochem Biophys Methods, 2000. 46(1-2): p. 69-81.
Bas, A., et al., Utility of the housekeeping genes 18S rRNA, beta-actin and glyceraldehyde-3-phosphate-dehydrogenase for normalization in real-time quantitative reverse transcriptase-polymerase chain reaction analysis of gene expression in human T lymphocytes. Scand J Immunol, 2004. 59(6): p. 566-73.
Catalan, V., et al., Validation of endogenous control genes in human adipose tissue: relevance to obesity and obesity-associated type 2 diabetes mellitus. Horm Metab Res, 2007. 39(7): p. 495-500.
Chechi, K., et al., Validation of Reference Genes for the Relative Quantification of Gene Expression in Human Epicardial Adipose Tissue. PLOS ONE, 2012. 7(4): p. e32265.
Zhang, W.X., et al., Selection of Suitable Reference Genes for Quantitative Real-Time PCR Normalization in Three Types of Rat Adipose Tissue. Int J Mol Sci, 2016. 17(6).
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