Real-time quantitative PCR analysis of rat embryonic stem cells (ESCs)

Validation study of a focused microarray for evaluation of undifferentiated rat ESCs.Using a focused microarray, undifferentiated embryonic stem cells (ESCs) can be distinguished from differentiated ESCs and other cells derived from the early embryo since they have a unique gene expression pattern associated with pluripotency and lack of markers of differentiation. To date, however, such an array has not been developed for the rat species and differences in genomes of rat and human or rat and mouse preclude the use of ESC focused human or mouse microarrays for the rat. Here, we developed a focused microarray for screening rat ESCs and provide validation data that this array can distinguish undifferentiated rat ESCs from rat trophoblast stem cells (TS), rat extraembryonic endoderm cells (XEN), mouse embryonic fibroblast feeder cells (MEFs) and rat ESCs that have been differentiated in vitro. We used this tool to compare rat ESCs which have been expanded in a conventional rat ESC medium containing two inhibitors, e.g., GSK3 and MEK inhibitors, and leukemia inhibitory factor (LIF), and found expression of Cdx2, a gene associated with trophoblast determination. The rat ESC focused microarray described in this report has utility for rapid screening rat ESCs. This will enable optimization of culture conditions in the future. A microarray was designed to compare the expression of 84 rat genes by various rat stem cell types and rat embryonic stem cell derived embryoid bodies. Relative fold changes in gene expression described by the microarray distinguish rat embryonic stem cells from other rat cell types, including rat embryonic stem cell derived embryoid bodies and mouse embryonic fibroblasts.