Abstract Background As the housekeeping genes (HKG) 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, HKG may vary gene expression profile according to different variables introducing systematic errors into experimental results. Sex bias can indeed affect expression display, however, up to date, 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 (AT) of Homo sapiens and Mus musculus and check sex bias and their overall suitability as internal controls. We also assess the expression stability of all genes included in distinct whole-transcriptome microarrays available from the Gene Expression Omnibus database to identify sex-unbiased housekeeping genes (suHKG) suitable for use as internal controls. We perform a novel computational strategy based on meta-analysis techniques to identify any sexual dimorphisms in mRNA expression stability in AT and to properly validate potential candidates. Results Just above half of the considered studies informed properly about the sex of the human samples, however, not enough female mouse samples were found to be included in this analysis. We found differences in the HKG expression stability in humans between female and male samples, with females presenting greater instability. We propose a suHKG signature including experimentally validated classical HKG like PPIA and RPL19 and novel potential markers for human AT and discarding others like the extensively used 18S gene due to a sex-based variability display in adipose tissue. Orthologs have also been assayed and proposed for mouse WAT suHKG signature. 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 research 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 . Highlights A computational strategy based on massive data analysis revealed that an accurate experimental design for adipose tissue requires the adequate selection of a sex-unbiased housekeeping genes (HKG). The extensively used 18S gene displays sex-based variability in adipose tissue, although PPIA and RPL19 do not, and hence, represent robust HKG. New sex-unbiased human and mouse candidate HKG: RPS8 and UBB. metafun-HKG ( https://bioinfo.cipf.es/metafun-HKG ): a freely available web tool to allow users to review stable expression levels of candidate HKG along the large volume of FAIR data.

Deciphering the Sex Bias in Housekeeping Gene Expression in Adipose Tissue: a Comprehensive Meta-analysis of Transcriptomic Studies