Hypoxanthine guanine phosphoribosyl transferase is the most stable reference gene for gene expression analysis by quantitative PCR in peripheral blood mononuclear cells from women with the polycystic ovary syndrome

Abstract

Background: The polycystic ovary syndrome (PCOS) is a frequent endocrine disorder that affects women of reproductive age. As the syndrome is strongly associated with obesity, it is of interest to examine the gene expression differences that accompany its development and the associated metabolic disturbances. Real-time RT PCR is a standard method for studying changes in gene expression. However, to obtain accurate and reliable results, validation of reference genes is obligatory. The aim of this study was to identify a suitable reference for the normalization of gene expression in peripheral blood mononuclear cells (PBMCs) from obese and normal-weight women with PCOS. Mathods: The expression stability of four potential reference genes: hypoxanthine guanine phosphoribosyl transferase 1 (HPRT), β-actin (BA), β2-microglobulin (B2M) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), was assessed in PBMCs from healthy women, and from normal-weight and obese women with PCOS. The variability in the expression of potential reference genes was analyzed by the TaqMan real-time RT PCR method, using GeNorm and NormFinder software packages. Results: Direct comparison of cycle threshold (Ct) values showed inter-individual variations for all validated genes, the Ct values of HPRT being less variable than those of BA, GAPDH and B2M. Both software packages pointed to HPRT as the most steadily expressed gene in the PBMCs of women with PCOS and healthy controls. Conclusions: Cross-validation of the expression stability of four potential reference genes identified HPRT as the most stable reference, suitable for further investigations of gene expression in PBMCs from women with PCOS.