Housekeeping gene transcript abundance in bovine fertilized and cloned embryos

The objective of this study was to compare housekeeping gene expression levels, relative to total mRNA, across different stages of bovine preimplantation development in embryos generated by IVF and somatic cell nuclear transfer (SCNT). We first analyzed the levels of total RNA recovered from different stages of preimplantation development. A similar RNA level was observed from oocytes to 16-cell stage embryos with a significant increase at morula and blastocyst stages. Then we used an absolute mRNA determination method that accounts for the RNA level in the embryo by quantifying copies of transcripts normalized to loaded cDNA amount. The number of housekeeping genes mRNA copies per nanogram of cDNA was compared among samples obtained from different stages of preimplantation IVF-derived embryos. None of the genes analyzed (GAPDH, PPIA, ACTB, RPL15, GUSB, and Histone H2A.2) maintained constant levels throughout preimplantation development, indicating that they are not suitable for normalizing gene expression across developmental stages. We then compared expression of housekeeping genes between IVF and SCNT embryos at different embryonic stages. We found different levels of transcript abundance between IVF and SCNT embryos for GAPDH, RPL15, GUSB, and ACTB. On the other hand, Histone H2A.2 and PPIA were similar between IVF and SCNT embryos at each stage analyzed, although they varied across stages as previously mentioned.