Assessing and validating housekeeping genes in normal, cancerous, and polycystic human ovaries
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REPRODUCTIVE PHYSIOLOGY AND DISEASE
Assessing and validating housekeeping genes in normal, cancerous, and polycystic human ovaries P. Asiabi 1 & J. Ambroise 2 C. A. Amorim 1
& C. Giachini
3
& M. E. Coccia
3
& B. Bearzatto
2
& M. C. Chiti
1
& M. M. Dolmans
1,4
&
Received: 31 March 2020 / Accepted: 23 July 2020 # Springer Science+Business Media, LLC, part of Springer Nature 2020
Abstract Purpose Housekeeping genes (HKGs), reference or endogenous control genes, are vital to normalize mRNA levels between different samples. Since using inappropriate HKGs can lead to unreliable results, selecting the proper ones is critical for gene expression studies. To this end, normal human ovaries, as well as those from patients diagnosed with ovarian endometrioid adenocarcinoma (OEA), ovarian mucinous adenocarcinoma (OMA), ovarian serous papillary carcinoma (OSPC), and polycystic ovary syndrome (PCOS), were used to identify the most suitable housekeeping genes. Methods RNA was isolated from 5 normal human ovaries (52–79 years of age), 9 cancerous ovaries (3 OEA, 3 OMA, 3 OSPC; 49–75 years of age), and 4 PCOS ovaries (18–35 years of age) in women undergoing hysterectomy. cDNA was synthesized using a whole transcriptome kit, and quantitative real-time PCR was performed using TaqMan array 96-well plates containing 32 human endogenous controls in triplicate. Results Among 32 HKGs studied, RPS17, RPL37A, PPIA, 18srRNA, B2M, RPLP0, RPLP30, HPRT1, POP4, CDKN1B, and ELF1 were selected as the best reference genes. Conclusions This study confirms recent investigations demonstrating that conventional HKGs, such as GAPDH and beta-actin, are not suitable reference genes for specific pathological conditions, emphasizing the importance of determining the best HKGs on a case-by-case basis and according to tissue type. Our results have identified reliable HKGs for studies of normal human ovaries and those affected by OEA, OMA, OSPC, or PCOS, as well as combined studies of control subjects vs. each cancer or PCOS group. Keywords Housekeeping/reference/endogenous control genes . Gene expression . Ovarian endometrioid adenocarcinoma (OEA) . Ovarian mucinous adenocarcinoma (OMA) . Ovarian serous papillary carcinoma (OSPC) . Polycystic ovary syndrome (PCOS)
Electronic supplementary material The online version of this article (https://doi.org/10.1007/s10815-020-01901-8) contains supplementary material, which is available to authorized users. * C. A. Amorim [email protected] 1
Pôle de Recherche en Gynécologie, Institut de Recherche Expérimentale et Clinique, Université Catholique de Louvain, Avenue Mounier 52, bte B1.52.02, 1200 Brussels, Belgium
2
Centre de Technologies Moléculaires Appliquées, Institut de Recherche Expérimentale et Clinique, Université Catholique de Louvain, 1200 Brussels, Belgium
3
Department of Clinical and Experimental Biomedical Sciences, University of Florence, 50134 Florence, Italy
4
Gynecology Department, Cliniques Universitaires Saint-Luc, 1200 Brussels, Belgium
Introduction Transcriptional re
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