Mammalian oocytes and embryos rely exclusively on maternal mRNAs to accomplish early developmental processes. Since oocytes and early embryos are transcriptionally silent after meiotic resumption, most of the synthesised maternal mRNA does not undergo immediate translation but is instead stored in the oocyte. Quantitative RT-PCR is commonly used to quantify mRNA levels, and correct quantification relies on reverse transcription and the choice of reference genes. Different methods for reverse transcription may affect gene expression determination in oocytes. In this study, we examined the suitability of either random or oligo(dT) primers for reverse transcription to be used for quantitative RT-PCR. We further looked for changes in poly(A) length of the maternal mRNAs during oocyte maturation. Our data indicate that depending on the method of reverse transcription, the optimal combination of reference genes for normalisation differed. Surprisingly, we observed a shortening of the poly(A) tail lengths of maternal mRNA as oocytes progressed from germinal vesicle to metaphase II. Overall, our findings suggest dynamic maternal regulation of mRNA structure and gene expression during oocyte maturation and early embryo development.

中文翻译：

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逆转录启动方法影响卵母细胞和早期胚胎中基因表达水平的标准化选择

哺乳动物卵母细胞和胚胎完全依赖母体 mRNA 来完成早期发育过程。由于卵母细胞和早期胚胎在减数分裂恢复后转录沉默，大多数合成的母体 mRNA 不会立即翻译，而是储存在卵母细胞中。定量 RT-PCR 通常用于量化 mRNA 水平，正确的量化依赖于逆转录和参考基因的选择。不同的逆转录方法可能会影响卵母细胞中的基因表达测定。在这项研究中，我们检查了随机或 oligo(dT) 引物是否适合用于定量 RT-PCR 的逆转录。我们进一步寻找在卵母细胞成熟过程中母体 mRNA 的 poly(A) 长度的变化。我们的数据表明，根据逆转录方法的不同，用于标准化的参考基因的最佳组合是不同的。令人惊讶的是，我们观察到随着卵母细胞从生泡发育到中期 II，母体 mRNA 的 poly(A) 尾长度缩短。总体而言，我们的研究结果表明，在卵母细胞成熟和早期胚胎发育过程中，母体对 mRNA 结构和基因表达的动态调节。