Reproduction Abstracts

Real-time RT quantitative PCR (qPCR) has become the most frequently used system for studies of gene expression. Many studies have provided reliable evidences that the transcription levels of reference genes are not constant at different developmental stages and in different experimental conditions. However, suitable reference genes which are stably expressed in polyploid preimplantation embryos of different developmental stages have not yet been identified. In the present study, we examined the expression levels of 12 candidate reference genes in preimplantation embryos of four different ploidies at six developmental stages. Stability analysis of the reference genes was performed by four independent software programs, namely, geNorm, NormFinder, the comparative ΔC_t method, and RefFinder. The stability of the selected three reference genes was evaluated by comparison with the Oct4 expression level during preimplantation development in diploid embryos. The expression levels of most genes in the polyploid embryos were higher than that in the diploid embryos, but increasing degree were disproportionate with the ploidies. There were no significant differences in reference genes expression among embryos of different ploidies when they reached the morula stage, and the expression level remained flat until the blastocyst stage. We conclude that Ubc, Ppia, and Pgk1 are the most stable reference genes for gene expression analysis of mouse diploid and polyploid preimplantation stage embryos.