Application of cDNA Arrays to monitor mRNA profiles in single preimplantation mouse embryos

摘要

Array technology is a widely used tool for gene expression profiling in various biological systems. However, the application of this method to mammalian preimplantation embryos is limited by the small amount of mRNA that can be extracted from a single embryo, which is not sufficient for array analysis. Here we report a protocol for the rapid global amplification of embryonic mRNA that hermits the generation of expression profiles from single murine blastocysts. The approach combines global PCR and T7 RNA polymerase amplification and allows the preparation of labeled, amplified RNA for array hybridization from single marine blastocysts containing approximately 1.5 pg mRNA in less than 12 h. We demonstrate that this amplification procedure is highly reproducible and does not bias original relative mRNA levels. Signal patterns from various embryonic stages of marine development revealed marked differences in mRNA expression that were in accordance with previously published data. We found genes known to be involved in embryonic apoptosis expressed at different levels in individual marine day 3.5 blastocysts. This technique can thus be used to assess embryonic viability, and investigate molecular mechanisms of embryonic development.