Duncan Francesca E, Schultz Richard M
Department of Obstetrics and Gynecology, Northwestern University, Chicago, Illinois, USA.
Methods Enzymol. 2010;477:457-80. doi: 10.1016/S0076-6879(10)77023-3.
Gene expression profiling using microarray technology is a robust, efficient, and cost-effective approach to assay a cell or tissue's transcriptome at a particular time or under a specific condition. Application of this technology to oocytes and preimplantation embryos has been limited largely because this biological material is difficult to acquire in sufficient quantities. We describe here a protocol to isolate and amplify mRNA from oocytes and preimplantation embryos that is suitable for microarray experiments. This protocol is based on a linear two-step amplification protocol using T7 RNA polymerase-based in vitro transcription and has been used to isolate more than 80 microg of cRNA from only 20 oocytes or preimplantation embryos. Gene expression profiling has provided insight into the molecular mechanisms of meiotic maturation, fertilization, and preimplantation embryo development. It has also been used to characterize female gametes and embryos from animals harboring gene-specific knockouts or knockdowns. Finally, this technology has been useful in evaluating how various Assisted Reproductive Technologies impact global patterns of gene expression in resulting embryos.
使用微阵列技术进行基因表达谱分析是一种强大、高效且经济有效的方法，可在特定时间或特定条件下检测细胞或组织的转录组。这项技术在卵母细胞和植入前胚胎中的应用受到很大限制，主要是因为这种生物材料难以获得足够的量。我们在此描述一种从卵母细胞和植入前胚胎中分离和扩增mRNA的方案，该方案适用于微阵列实验。该方案基于使用基于T7 RNA聚合酶的体外转录的线性两步扩增方案，并且已用于仅从20个卵母细胞或植入前胚胎中分离出超过80微克的cRNA。基因表达谱分析为减数分裂成熟、受精和植入前胚胎发育的分子机制提供了深入了解。它还被用于表征来自具有基因特异性敲除或敲低的动物的雌配子和胚胎。最后，这项技术有助于评估各种辅助生殖技术如何影响所得胚胎中基因表达的全局模式。
