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.

中文翻译：

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评估和验证正常、癌变和多囊人类卵巢中的管家基因。

目的

管家基因 (HKG)、参考基因或内源性对照基因对于标准化不同样品之间的 mRNA 水平至关重要。由于使用不合适的 HKG 会导致不可靠的结果，因此选择合适的 HKG 对基因表达研究至关重要。为此，使用正常人类卵巢以及诊断为卵巢子宫内膜样腺癌 (OEA)、卵巢粘液性腺癌 (OMA)、卵巢浆液性乳头状癌 (OSPC) 和多囊卵巢综合征 (PCOS) 的患者的卵巢来识别最合适的看家基因。

方法

从 5 个正常人类卵巢（52-79 岁）、9 个癌性卵巢（3 个 OEA、3 个 OMA、3 个 OSPC；49-75 岁）和 4 个 PCOS 卵巢（18-35 岁）中分离出 RNA在接受子宫切除术的女性中。使用全转录组试剂盒合成 cDNA，并使用包含 32 个人内源对照一式三份的 TaqMan 阵列 96 孔板进行实时定量 PCR。

结果

在研究的 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。

结论

这项研究证实了最近的研究，表明传统的 HKG，如 GAPDH 和β-肌动蛋白，不是特定病理条件的合适参考基因，强调了根据具体情况和组织类型确定最佳 HKG 的重要性。我们的结果确定了可靠的 HKG，可用于研究正常人类卵巢和受 OEA、OMA、OSPC 或 PCOS 影响的卵巢，以及对照受试者与每个癌症或 PCOS 组的联合研究。