The relationship between the conceptus and the maternal uterine environment is crucial for the successful establishment and maintenance of pregnancy in cattle. Gene expression analysis of the conceptus and maternal reproductive tissues is a favourable method to assess the embryonic maternal interaction. The reliability of the commonly used method reverse transcription‐quantitative polymerase chain reaction (RT‐qPCR) depends on proper normalization to stable reference genes (RGs). The objective of this study was to determine the expression stability of 10 potential RGs in maternal reproductive tissues and foetal tissues, and to analyse the effect of RG selection on the calculation of the relative expression of target genes. The expression stability of 10 potential RGs was analysed in eight different tissues from three pregnant dairy cows. Three programs—GeNorm, NormFinder and Bestkeeper—were used to identify the best RGs. According to all three programs, the most stable RG was CNOT11, whereas the least stable RGs were GAPDH and HPRT1. GeNorm analysis showed that a combination of five RGs (SDHA, PPIA, CNOT11, RPS9 and RPL19) was necessary for appropriate data normalization. However, NormFinder analysis indicated that the combination of CNOT11 and PPIA was the most suitable. When target genes were normalized to these RGs, the relative expression of the Radical S‐adenosyl methionine domain containing 2 gene was not affected by the choice of RGs, whereas a large difference was observed in the expression profile of the Nuclear erythroid2‐related factor 2 gene between the most stable and least stable RGs. The results indicate that careful selection of RGs is crucial under different conditions, especially for target genes with relatively small fold changes. Furthermore, the results provide useful information for the selection of RGs for evaluating genes affecting bovine reproduction.

中文翻译：

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鉴定可靠的参考基因，用于在怀孕母牛的母体生殖组织和胎儿组织中进行表达研究。

妊娠与母体子宫环境之间的关系对于牛的成功建立和维持妊娠至关重要。概念和母体生殖组织的基因表达分析是评估胚胎母体相互作用的一种有利方法。常用方法逆转录定量聚合酶链反应（RT-qPCR）的可靠性取决于对稳定参考基因（RGs）的正确标准化。本研究的目的是确定10种潜在RGs在母体生殖组织和胎儿组织中的表达稳定性，并分析RG选择对靶基因相对表达计算的影响。分析了来自三只怀孕奶牛的八种不同组织中10种潜在RGs的表达稳定性。使用三个程序（GeNorm，NormFinder和Bestkeeper）来确定最佳RG。根据这三个程序，最稳定的RG是CNOT11，而最不稳定的RGs是GAPDH和HPRT1。GeNorm分析表明，五个RG（SDHA，PPIA，CNOT11，RPS9和RPL19）的组合对于适当的数据标准化是必要的。但是，NormFinder分析表明，CNOT11和PPIA的组合是最合适的。当靶基因被标准化到这些RG时，自由基S的相对表达包含2个基因的腺苷甲硫氨酸域不受RGs选择的影响，而在最稳定和最不稳定的RGs之间，在核红细胞相关因子2基因的表达谱中观察到了很大的差异。结果表明，在不同条件下仔细选择RGs至关重要，尤其是对于倍数变化相对较小的靶基因。此外，结果为选择RGs以评估影响牛繁殖的基因提供了有用的信息。