Non-radioisotopic quantitative RT-PCR to detect changes in mRNA levels during early mouse embryo development.
Non-radioisotopic quantitative RT-PCR to detect changes in mRNA levels during early mouse embryo development.
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DOI：
10.1006/bbrc.1993.2112
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发表时间：
1993-09
影响因子：
3.1
通讯作者：
H. Yokoi;S. Natsuyama;M. Iwai;Y. Noda;T. Mori;Kazuhiro J. Mori;K. Fujita;H. Nakayama;J. Fujita
中科院分区：
文献类型：
--
作者：
H. Yokoi;S. Natsuyama;M. Iwai;Y. Noda;T. Mori;Kazuhiro J. Mori;K. Fujita;H. Nakayama;J. Fujita
我们建立了一种灵敏度高、重现性好的非放射性同位素定量RT-PCR方法。该RT-PCR结果与Northern blot分析结果一致。我们采用RT-PCR方法检测了早期小鼠胚胎中β -肌动蛋白、转铁蛋白受体和两个细胞周期相关基因cyclin B和cdc25的mRNA水平。在两细胞期晚期胚胎中，β -肌动蛋白、转铁蛋白受体和细胞周期蛋白B mRNA水平是MII期卵母细胞的10-20%。相比之下，cdc25 mRNA水平在这些阶段之间没有差异。当我们培养小鼠胚胎时，培养基中存在RNA聚合酶抑制剂α -amanitin并不影响双细胞阶段的mRNA水平，这表明在双细胞胚胎中检测到的大多数mRNA都是母源的。这些结果表明，细胞周期蛋白B和cdc25在胚胎早期的mRNA降解速率是不同的。
We developed a non-radioisotopic quantitative RT-PCR method with high sensitivity and reproducibility. The results of this RT-PCR were in agreement with those of the Northern blot analysis. We measured the mRNA levels of beta-actin, transferrin receptor, and two cell cycle-related genes, cyclin B and cdc25, in early mouse embryos by the RT-PCR. In late two-cell stage embryos, beta-actin, transferrin receptor and cyclin B mRNA levels were 10-20% of those in MII stage oocytes. In contrast, the cdc25 mRNA levels were not different between these stages. When we cultured mouse embryos, the presence of an RNA polymerase inhibitor, alpha-amanitin, in the medium did not affect the mRNA levels at the two-cell stage, indicating that most of the detected mRNAs in two-cell embryos were maternally derived. These results suggest that the rate of mRNA degradation is different between cyclin B and cdc25 during early embryogenesis.