A hallmark of biology is the cyclical nature of organismal physiology driven by networks of biological, including circadian, rhythms. Unsurprisingly, disruptions of the circadian rhythms through sleep curtailment or shift work have been connected through numerous studies to positive associations with obesity, insulin resistance, and diabetes. Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) measures oscillation in messenger RNA expression, an essential foundation for the study of the physiological circadian regulatory network. Primarily, measured oscillations have involved the use of reference gene normalization. However, the validation and identification of suitable reference genes is a significant challenge across different biological systems. This study focuses on adipose tissue of premenopausal, otherwise healthy, morbidly obese women voluntarily enrolled after being scheduled for laparoscopic sleeve gastrectomy surgery. Acquisition of tissue was accomplished by aspiratory needle biopsies of subcutaneous adipose tissue 1 to 2 weeks prior to surgery and 12 to 13 weeks following surgery and an in-surgery scalpel-assisted excision of mesenteric adipose tissue. Each biopsy was sterile cultured ex vivo and serially collected every 4 h over approximately 36 h. The candidate reference genes that were tested were 18S rRNA, GAPDH, HPRT1, RPII, RPL13α, and YWHAZ. Three analytic tools were used to test suitability, and the candidate reference genes were used to measure oscillation in expression of a known circadian clock element (Dbp). No gene was deemed suitable as an individual reference gene control, which indicated that the optimal reference gene set was the geometrically averaged 3-gene panel composed of YWHAZ, RPL13α, and GAPDH. These methods can be employed to identify optimal reference genes in other systems.

中文翻译：

---

人类脂肪组织昼夜节律基因表达分析的参考基因优化。


生物学的一个标志是由生物网络（包括昼夜节律）驱动的有机体生理学的周期性。毫不奇怪，大量研究表明，由于睡眠减少或轮班工作而造成的昼夜节律紊乱与肥胖、胰岛素抵抗和糖尿病呈正相关。定量逆转录聚合酶链式反应 (qRT-PCR) 可测量信使 RNA 表达的振荡，这是研究生理昼夜节律调节网络的重要基础。首先，测量的振荡涉及参考基因标准化的使用。然而，合适的参考基因的验证和鉴定是不同生物系统中的重大挑战。这项研究的重点是绝经前健康的病态肥胖女性的脂肪组织，这些女性在计划进行腹腔镜袖状胃切除手术后自愿加入。通过手术前 1 至 2 周和手术后 12 至 13 周对皮下脂肪组织进行抽吸性针活检以及手术中手术刀辅助切除肠系膜脂肪组织来获取组织。每个活组织检查均经过无菌离体培养，并在大约 36 小时内每 4 小时连续收集一次。测试的候选参考基因是 18S rRNA、GAPDH、HPRT1、RPII、RPL13α 和 YWHAZ。使用三种分析工具来测试适用性，并使用候选参考基因来测量已知生物钟元件 (Dbp) 表达的振荡。没有基因被认为适合作为单个参考基因对照，这表明最佳参考基因集是由YWHAZ、RPL13α和GAPDH组成的几何平均3基因组。 这些方法可用于识别其他系统中的最佳参考基因。