Validation of reference genes for RT-qPCR in in vitro cat oocytes and preimplantation embryos

Quantitative real-time PCR (qPCR) is a commonly used approach for accurate assessment of gene transcription patterns in oocytes and early embryos. Comparing gene expression across different samples can lead to erroneous conclusions due to differences in amount of starting material, reverse-transcription yield or enzymatic efficiencies. An elegant way to control for these variables is normalisation by using the geometric mean of the most stable reference genes as an internal control. The stability of reference genes differs between species and tissue type. In this study, a set of reliable reference genes was selected for cat oocytes and early embryos. Oocytes were in vitro matured, fertilized, cultured and collected for RNA extraction and DNAse treatment, followed by conversion into cDNA. Transcription levels of 10 commonly used reference genes (ACTB, GAPDH, SDHA, RPS7, RPL17, HMBS, HPRT, GUSB, YWHAZ and B2M) were determined in single oocytes (n = 20) and embryos at different reimplantation stages (8-cell, compact morula, blastocyst, hatching blastocyst, n = 10/stage) using previously optimized qPCR programs. For each gene, qPCR was performed on all samples in duplex along with a dilution series in the same run. All data were interpreted using geNorm software (identifying YWHAZ, SDHA and GUSB as the most stable genes during early development, while RPS7, RPL30 and GAPDH appeared to be highly regulated (http://medgen.ugent.be/~jvdesomp/genorm), Based on these results, the geometric mean of YWHAZ, SDHA and GUSB is suggested for appropriate normalization of RTqPCR studies of cat oocytes and early embryos