Simple and efficient transcriptome profiling from a single embryo. Mus musculus

Measuring the global gene expression pattern in a single cell has been technically challenging, but is potentially very useful for understanding variation in biological processes between cells and for studying gene regulation during early embryo development with single cell resolution. To advance these applications, multiple systems for single-cell RNA extraction in addition to crude cell lysis followed by RNA profiling were examined and used for genomic data analysis. Our results indicate that some of these methods are suitable for analyzing even a portion of the total RNA from a single oocyte or embryo, with low variance among technical replicates across a wide dynamic range of expression. These methods will not only be helpful for genomic and epigenetic research to identify regulatory mechanisms of early development and molecular signatures of embryo classes, but can also provide great potential for clinical analysis of small biopsy samples or pre-implantation genetic disease screening. Overall design: RNA from single mouse oocytes was purified using Qiagen Rneasy Mini or Arcturus PicoPure kits. Crude lysates of single oocytes were also prepared in NuGEN direct lysis buffer. Three replicate oocytes for each of the three sample preparation methods were used. Total RNA was amplified by NuGEN Ovation One-Direct ribo-SPIA, cDNA products were biotinylated, and labeled targets were hybridized to Affymetrix Mouse Gene 1.0ST Arrays.