Assessing and validating housekeeping genes in normal, cancerous, and polycystic human ovaries

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) * C. A.

Validating Reference Gene Expression Stability in Human Ovarian Follicles, Oocytes, Cumulus Cells, Ovarian Medulla, and Ovarian Cortex Tissue

“…Our results are comparable to the effect of how serum supplementation affects RG expression during culture in colon adenocarcinomas cell lines [15]. A few studies have also validated RG expression stability in biopsies from normal, cancerous, and polycystic ovaries (PCO) [10,[24][25][26] or pieces of cryopreserved versus the non-cryopreserved ovarian cortex [16], with variable outcomes. GAPDH has proven to be stably expressed in normal ovarian tissues [24], whereas both GAPDH and ACTB have been poorly ranked in PCO and cancerous ovarian tissue [10,25,26].…”

“…The NormFinder tool found ACTB to be the most stable gene for human immature oocytes and cumulus cells, and B2M to be the most stable gene for medulla tissue and isolated preantral follicles, provided a normalized RNA concentration was used. Even though ACTB is one of the most used RGs, some studies omit this as RG because its expression can be affected by biochemical stimuli, during cell growth and differentiation, or even by certain pathologies in various human cells and tissues [12], including ovarian tissue [10]. Nevertheless, our data suggest that ACTB expression in human immature oocytes is more stable than in other cell types as we obtained similar results with both fresh and in vitro cultured immature oocytes (data not shown).…”

“…A few studies have also validated RG expression stability in biopsies from normal, cancerous, and polycystic ovaries (PCO) [10,[24][25][26] or pieces of cryopreserved versus the non-cryopreserved ovarian cortex [16], with variable outcomes. GAPDH has proven to be stably expressed in normal ovarian tissues [24], whereas both GAPDH and ACTB have been poorly ranked in PCO and cancerous ovarian tissue [10,25,26]. It is worth noting that, in our study, the patient effect was considered within each treatment.…”

“…RGs are used to correct sample-to-sample variations, including the amount and integrity of RNA, enzymatic efficiency, and presence of contaminating DNA [8,9]. Ideally, RGs should be universally expressed with a constant level in any cell type and biological or experimental condition [10]. However, no universal RG has been detected so far.…”

“…However, most studies use a single RG to normalize RT-qPCR data without any preliminary evaluation of appropriateness [4,5,23]. Access to human ovarian tissue is very limited, especially oocytes, and only a few studies have validated RGs in biopsies from normal, cancerous, and/or polycystic ovaries [10,[24][25][26], or pieces of cryopreserved vs. non-cryopreserved ovarian cortex [16]. None of these previous studies have investigated RG expression stability in specific ovarian compartments, including oocytes, preantral follicles, cumulus cells, and medulla tissue.…”