Assessment of Sex Bias in Housekeeping Gene Expression in Adipose Tissue Through the Massive Analysis of Transcriptomics Data

Abstract Background: As the housekeeping genes generally involved in maintaining essential cell functions are typically assumed to exhibit constant expression levels across cell types, they are commonly employed as internal controls in gene expression studies. Nevertheless, multiple studies indicate that not all housekeeping genes display stable expression across cells and tissues and under various healthy and diseased conditions, which can introduce systematic errors into experimental results. The selection and validation of housekeeping genes as controls for each studied condition represent crucial steps in ensuring the validity of obtained results; however, up till now, sex has not been typically considered as a biological variable.Methods: In this study, we evaluate the expression profiles of six classical housekeeping genes (four metabolic: GAPDH, HPRT, PPIA, and UBC, and two ribosomal: 18S and RPL19) to determine expression stability in adipose tissues of Homo sapiens and Mus musculus and check sex bias and their overall suitability as internal controls. We also assess the stabile expression of genes included in distinct whole-transcriptome microarrays available from the Gene Expression Omnibus database to identify sex-unbiased housekeeping genes suitable for use as internal controls. We perform a novel computational strategy based on meta-analysis techniques to describe any sexual dimorphisms in mRNA expression stability.Results: Just above half of the considered studies informed properly about the sex of the samples. Found differences in the HKG expression stability in humans between female and male samples, with females presenting greater instability. The extensively used 18S gene displays sex-based variability in adipose tissue, although PPIA and RPL19 do not, and hence, represent robust housekeeping genes. All results generated during this study are readily available by accessing an open web resource (https://bioinfo.cipf.es/metafun-HKG) for consultation and reuse in further studies.Conclusions: This sex-based analysis proves that certain classical housekeeping genes fail to function adequately as controls when analyzing human adipose tissue considering sex as a variable. We confirm RPL19 and PPIA suitability as sex-unbiased human and mouse housekeeping genes derived from sex-specific expression profiles, and propose new ones such as RPS8 and UBB.