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.
Centre de Technologies Moléculaires Appliquées, Institut de Recherche Expérimentale et Clinique, Université Catholique de Louvain, 1200, Brussels, Belgium.
J Assist Reprod Genet. 2020 Oct;37(10):2545-2553. doi: 10.1007/s10815-020-01901-8. Epub 2020 Jul 30.
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.
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.
Among 32 HKGs studied, RPS17, RPL37A, PPIA, 18srRNA, B2M, RPLP0, RPLP30, HPRT1, POP4, CDKN1B, and ELF1 were selected as the best reference genes.
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.
管家基因（HKGs），即参考或内参基因，对于标准化不同样本之间的 mRNA 水平至关重要。由于使用不合适的 HKG 会导致结果不可靠，因此选择合适的 HKG 对于基因表达研究至关重要。为此，我们使用正常人类卵巢以及经诊断患有卵巢子宫内膜样腺癌（OEA）、卵巢黏液性囊腺癌（OMA）、卵巢浆液性乳头状癌（OSPC）和多囊卵巢综合征（PCOS）的卵巢组织，来鉴定最适合的管家基因。
从 5 例接受子宫切除术的正常女性（52-79 岁）、9 例癌症患者（3 例 OEA、3 例 OMA、3 例 OSPC；49-75 岁）和 4 例 PCOS 患者（18-35 岁）的卵巢中提取 RNA。使用全转录组试剂盒合成 cDNA，并使用 TaqMan 阵列 96 孔板进行定量实时 PCR，该板包含 32 个人内源对照基因，每个基因重复 3 次。
在所研究的 32 个 HKG 中，RPS17、RPL37A、PPIA、18srRNA、B2M、RPLP0、RPLP30、HPRT1、POP4、CDKN1B 和 ELF1 被选为最佳参考基因。
本研究证实了最近的研究结果，即传统的 HKG 如 GAPDH 和 beta-actin 不适合特定病理条件下的参考基因，强调了根据具体情况和组织类型确定最佳 HKG 的重要性。我们的研究结果为正常人类卵巢和受 OEA、OMA、OSPC 或 PCOS 影响的卵巢以及对照与每个癌症或 PCOS 组的联合研究确定了可靠的 HKG。
