Quantitative Evaluation of Reference Genes for Real‐Time PCR During In Vitro Maturation of Ovine Oocytes

Contents Quantitative reverse transcription PCR ( RT ‐ qPCR ) is a powerful molecular technique that enables gene expression studies to be performed on extremely small samples such as oocytes and pre‐implantation embryos. However, the high sensitivity of this technique requires that to prevent bias in expression data interpretation, the reference genes used for normalization are fully validated before use. Difficulties in choosing a reference gene are further compounded by the variable RNA content of the maturing oocyte. In the present study, we evaluated eight commonly used reference genes such as ACTB , GAPDH , H2AFZ , HPRT 1, PPIA , SDHA , TUBB and YWHAZ and for sheep oocyte RT ‐q PCR before and after in vitro maturation. We have also compared different c DNA priming strategies using random hexamers or oligo‐d T . Ge N orm analysis of the results identified the most reliable genes for normalization to be SDHA , TUBB and PPIA when oocyte c DNA was made with random hexamers, and YWHAZ , TUBB and SDHA when oligo‐d T primers were used ( H 2 AFZ and HPRT 1 were excluded from the ge N orm analysis). Interestingly, the analysis revealed that the least stable genes were ACTB and GAPDH , which are the conventional ‘housekeeping’ genes used in many studies. We recommend the use of three reference genes to calculate a normalization factor to accurately quantify transcript abundance in sheep oocytes and these vary with the c DNA priming strategy employed.