Introduction of Reference Genes for Quantitative Real-Time PCR Data Normalization in Cryopreserved Stallion Sperm

Cryopreservation has led to many improvements in domestic animal breeding industry, especially in terms of spreading of superior genes. The improvement of extenders and cryoprotectants can result in a significant increase in transporting and inseminating of frozen stallion semen. In this study, we analysed the genes that would be expressed in stallion cryopreserved sperm and by providing an adequate amplification, they would be selected as reference genes in the future studies. Live sperm with above 70% motility were selected from six different stallions. Sperm samples were divided in to two parts for total mRNA extraction. The selective amplification of three candidate genes (L32, βactin and Ubiquitin) were transcribed by real-time polymerase chain reaction (RT-qPCR). Gene stabilities were calculated by GeNorm and BestKeeper softwares. Results indicated that the expression of L32 gene is less stable than that of β-actin and Ubiquitin. The expression stability of β-actin and Ubiquitin were similar. These genes can be used for normalizing of RT-qPCR reaction data. The GeNorm software introduced the best combination of genes as, β-actin and Ubiquitin with the stability value (M) of 0.083. The results of BestKeeper software showed that the most stable house-keeping gene was β-actin.