Ge Caiyun, Yu Pengxia, Fang Man, Wang Hui, Zhang Yuanzhen
Department of Obstetrics and Gynaecology, Zhongnan Hospital of Wuhan University, 169 Donghu Road, Wuchang District, Wuhan, 430071, China.
Department of Pharmacology, Basic Medical School of Wuhan University, 185 Donghu Road, Wuchang District, Wuhan, 430071, China.
Mol Cell Biochem. 2021 Jul;476(7):2613-2622. doi: 10.1007/s11010-021-04115-3. Epub 2021 Mar 3.
The selection of suitable reference genes (RGs), especially the identification of the proper combination of RGs is the key to obtain reliable results of gene expression for quantitative real-time polymerase chain reaction (qRT-PCR). To date, there is no relevant study dealing with the stability of RGs in rat placenta. In this study, the geNorm, NormFinder, and BestKeeper software were used to analyze the expression stability of the candidate RGs in placenta under physiological and prenatal caffeine exposure (PCE) conditions. The expression of Tbp, Gapdh and Ywhaz in female and Polr2a, Gapdh and Ywhaz in male placenta were highly stable under physiological conditions, and there was no obvious gender difference. We further found that two RGs were sufficient for reliable normalization in female and male placenta and the combination of Ywhaz and Gapdh was the most suitable compound RGs under physiological conditions. Under PCE conditions, Ywhaz, Gapdh and Polr2a were the most stable genes in both female and male placenta. Among them, Ywhaz and Gapdh were chosen as the best paring. Finally, selected RGs were employed for normalization of the expression of a clear target gene and the results of standardization supported our choice. In conclusion, our study confirmed that Ywhaz/Gapdh combination was the most suitable RGs in rat placenta under physiological and PCE pathological conditions and provided a theoretical and experimental basis for physiological and pathological research of the rat placenta.
选择合适的内参基因（RGs），尤其是确定RGs的恰当组合，是通过定量实时聚合酶链反应（qRT-PCR）获得可靠基因表达结果的关键。迄今为止，尚无关于大鼠胎盘内参基因稳定性的相关研究。在本研究中，使用geNorm、NormFinder和BestKeeper软件分析了在生理条件和产前咖啡因暴露（PCE）条件下候选内参基因在胎盘中的表达稳定性。在生理条件下，雌性胎盘中Tbp、Gapdh和Ywhaz以及雄性胎盘中Polr2a、Gapdh和Ywhaz的表达高度稳定，且无明显性别差异。我们进一步发现，两个内参基因足以在雌性和雄性胎盘中进行可靠的标准化，并且在生理条件下，Ywhaz和Gapdh的组合是最合适的复合内参基因。在PCE条件下，Ywhaz、Gapdh和Polr2a是雌性和雄性胎盘中最稳定的基因。其中，Ywhaz和Gapdh被选为最佳配对。最后，使用选定的内参基因对一个明确的靶基因的表达进行标准化，标准化结果支持了我们的选择。总之，我们的研究证实，在生理和PCE病理条件下，Ywhaz/Gapdh组合是大鼠胎盘中最合适的内参基因，为大鼠胎盘的生理和病理研究提供了理论和实验依据。
