Abstract
Comparative gene expression analysis by qRT-PCR is commonly used to detect differentially expressed genes in studies of PCOS pathology. Impaired GC function is strongly associated with PCOS pathogenesis, and a growing body of studies has been dedicated to identifying differentially expressed genes in GCs in PCOS patients and healthy women by qRT-PCR. It is necessary to validate the expression stability of the selected reference genes across the tested samples for target gene expression normalization. We examined the variability and stability of expression of the 15 commonly used reference genes in GCs from 44 PCOS patients and 45 healthy women using the GeNorm, BestKeeper, and NormFinder statistical algorithms. We combined the rankings of the three programs to produce a final ranking based on the geometric means of their stability scores. We found that HPRT1, RPLP0, and HMBS out of 15 examined commonly used reference genes are stably expressed in GCs in both controls and PCOS patients and can be used for normalization in gene expression profiling by qRT-PCR. Future gene-expression studies should consider using these reference genes in GCs in PCOS patients for more accurate quantitation of target gene expression and data interpretation.
Highlights
Polycystic ovarian syndrome (PCOS) is characterized by ovulatory dysfunction, polycystic ovaries, and hyperandrogenism and is a common gynecological endocrinopathy and a leading cause of female reproductive failure[1, 2]
Given the crucial role of granulosa cells (GCs) in PCOS pathology, we examined the expression stability and variability of the following 15 commonly used reference genes: RNA18S5, ACTB, GAPDH, ubiquitin C (UBC), β-2-microglobulin (B2M), RNA polymerase II subunit A (POLR2A), hypoxanthine phosphoribosyltransferase 1 (HPRT1), ζ-tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein (YWHAZ), hydroxymethylbilane synthase (HMBS), β-glucuronidase (GUSB), importin 8 (IPO8), phosphoglycerate kinase 1 (PGK1), ribosomal protein lateral stalk subunit P0 (RPLP0), transferrin receptor (TFRC), and ribosomal protein L13a (RPL13A) in GCs from healthy control subjects and PCOS patients
The body mass index (BMI) of the PCOS group was significantly higher than the control group by 7.8% (PCOS: 24.20 ± 4.73 kg/m2, control: 22.45 ± 2.99 kg/m2, 95% CI: −1.771 to 9.411, t = 1.358 df = 87, p < 0.05)
Summary
Polycystic ovarian syndrome (PCOS) is characterized by ovulatory dysfunction, polycystic ovaries, and hyperandrogenism and is a common gynecological endocrinopathy and a leading cause of female reproductive failure[1, 2]. To the best of our knowledge, there are no published data that can be used to select reliable reference genes for gene expression normalization in the GCs of reproductively healthy women and PCOS patients. Given the crucial role of GCs in PCOS pathology, we examined the expression stability and variability of the following 15 commonly used reference genes: RNA18S5, ACTB, GAPDH, ubiquitin C (UBC), β-2-microglobulin (B2M), RNA polymerase II subunit A (POLR2A), hypoxanthine phosphoribosyltransferase 1 (HPRT1), ζ-tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein (YWHAZ), hydroxymethylbilane synthase (HMBS), β-glucuronidase (GUSB), importin 8 (IPO8), phosphoglycerate kinase 1 (PGK1), ribosomal protein lateral stalk subunit P0 (RPLP0), transferrin receptor (TFRC), and ribosomal protein L13a (RPL13A) in GCs from healthy control subjects and PCOS patients. This study provides valuable insights into the selection of appropriate reference genes for gene expression profiling in GCs from PCOS patients
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
