Quantitative analysis of messenger RNA abundance for ribosomal protein L-15, cyclophilin-A, phosphoglycerokinase, β-glucuronidase, glyceraldehyde 3-phosphate dehydrogenase, β-actin, and histone H2A during bovine oocyte maturation and early embryogenesis in vitro
Quantitative analysis of messenger RNA abundance for ribosomal protein L-15, cyclophilin-A, phosphoglycerokinase, β-glucuronidase, glyceraldehyde 3-phosphate dehydrogenase, β-actin, and histone H2A during bovine oocyte maturation and early embryogenesis in vitro
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DOI：
10.1002/mrd.20333
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发表时间：
2006-03-01
影响因子：
2.5
通讯作者：
Smith, GW
中科院分区：
文献类型：
--
作者：
Bettegowda, A;Patel, OV;Smith, GW
实时逆转录PCR大大提高了定量基因表达研究的简便性和灵敏度。然而，基因表达的测量通常需要选择用于数据标准化的有效参考（管家基因）以补偿固有变化。鉴于早期胚胎发育的动态性质，将该技术应用于卵母细胞和早期胚胎发育的研究进一步复杂化，这是由于起始材料的量有限以及用于数据标准化的组成型表达基因的信息缺乏。我们已经验证了定量程序实时逆转录聚合酶链反应（RT-PCR）分析的mRNA丰度在牛减数分裂成熟和早期胚胎发生，并利用这种技术来确定核糖体蛋白L-15，亲环蛋白-A，磷酸甘油激酶，β-葡萄糖醛酸酶，甘油醛-3-磷酸脱氢酶，β-肌动蛋白和组蛋白H2 A的mRNA丰度的时间变化。定量的特定外源RNA添加到样品中的量显示了可接受的RNA回收率和逆转录效率，具有最小的变化。牛卵母细胞发育至中期11导致多聚腺苷酸化丰度降低，但上述大多数基因的总转录物未降低;然而，磷酸甘油激酶在两种RNA群体中均表现出显著下降。上述基因mRNA在早期胚胎中的表达量一直较低，直至囊胚期，但在桑椹胚期核糖体蛋白L-15 mRNA的表达量增加，组蛋白H2 A mRNA在胚胎基因组激活前呈现动态变化。结果表明，一个有效的方法来定量分析mRNA丰度在卵母细胞和胚胎，但不支持上述基因的组成型表达在早期胚胎发育。
Real-time reverse transcription PCR has greatly improved the ease and sensitivity of quantitative gene expression studies. However, measurement of gene expression generally requires selection of a valid reference (housekeeping gene) for data normalization to compensate for inherent variations. Given the dynamic nature of early embryonic development, application of this technology to studies of oocyte and early embryonic development is further complicated due to limited amounts of starting material and a paucity of information on constitutively expressed genes for data normalization. We have validated quantitative procedures for real-time reverse transcription polymerase chain reaction (RT-PCR) analysis of mRNA abundance during bovine meiotic maturation and early embryogenesis and utilized this technology to determine temporal changes in mRNA abundance for ribosomal protein L-15, cyclophilin-A, phosphoglycerokinase, beta-glucuronidase, glyceraldehyde-3-phosphate dehydrogenase, beta-actin, and histone H2A. Quantification of amounts of specific exogenous RNAs added to samples revealed acceptable rates of RNA recovery and efficiency of reverse transcription with minimal variation. Progression of bovine oocytes to metaphase 11 resulted in reduced abundance of polyadenylated, but not total transcripts for majority of above genes; however phosphoglycerokinase exhibited a significant decline in both RNA populations. Abundance of mRNAs for above genes in early embryos generally remained low until the blastocyst stage, but abundance of ribosomal protein L-15 mRNA was increased at the morula stage and histone H2A mRNA showed dynamic changes prior to embryonic genome activation. Results demonstrate a valid approach for quantitative analysis of mRNA abundance in oocytes and embryos, but do not support constitutive expression of above genes during early embryonic development.