Sex bias evaluation of classic and novel Housekeeping Genes in adipose tissue through the massive analysis of transcriptomics data

Housekeeping genes (HKG), those involved in the maintenance of basic cell functions, are considered to have constant expression levels in all cell types, and are therefore commonly used as internal controls in gene expression studies. Nevertheless, multiple studies have shown that not all of them have stable expression levels across different cells, tissues, and conditions, introducing a systematic error in the experimental results. The proper selection and validation of control housekeeping genes in the specific studied conditions is crucial for the validity of the obtained results, although, up to date, sex has never been taken into account as a biological variable. In this work, we evaluate the expression profiles of six classical housekeeping genes, (four metabolic: HPRT , GAPDH , PPIA and UBC , and two ribosomal: 18S and RPL19 ) used as controls in several tissues, to determine the stability of their expression in adipose tissue of Homo sapiens and Mus musculus and asses sex bias and control suitability. We also evaluated gene expression stability of the genes included in different whole transcriptome microarrays available at the Gene Expression Omnibus database (GEO), to identify new genes suitable to be used as sex-unbiased controls. We perform a sex-based analysis to test for/reveal sexual dimorphism of mRNA expression stability.

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