Identification of Stable Reference Genes for Gene Expression Studies Using Quantitative Real Time PCR in Buffalo Oocytes and Embryos


Author’s address (for correspondence): Tirtha Kumar Datta, Animal Genomics Lab, Animal Biotechnology Centre, National Dairy Research Institute, Karnal, Haryana 132001, India. E-mail:


The present study was aimed to validate expression stability of 6 housekeeping genes (viz. YWHAZ, SDHA, GAPDH, RPS15, RPS18 and RN18S1) in the oocytes and embryos of different stages in buffalo. A modified Trizol protocol was optimized for RNA isolation from as few as five oocytes. The expression level of selected genes was studied by an optimized real time PCR using DCT method and their stability of expression was evaluated by Microsoft Excel based visual application, geNORM. The analysis revealed that the RPS15 and GAPDH were the most stable genes across different samples. Also, the geometric mean of three genes (i.e. RPS15, RPS18 and GAPDH) were found suitable for normalization of real time PCR data from buffalo oocytes/embryos. The information would help in more accurate interpretation of gene expression data from oocytes/embryos towards understanding the molecular events in these cells during development.