Development and optimization of an efficient RNA isolation protocol from bovine (Bos indicus) spermatozoa

Achieving the optimum extraction of RNA from spermatozoal cells is crucial for carrying out effective high-throughput analysis regarding its role in fertility and other reproduction processes in Bos indicus. Nevertheless, semen comprises spermatozoa and several other secretions from the male reproductive system, which as well consist of diverse somatic cell types. Therefore, the elimination of somatic cells guarantees the purity of the sperm RNA. In the present study, we tested five different RNA isolation protocols and evaluated them for their yield and purity using spectrophotometer and polymerase chain reaction. Among the five RNA isolation protocols, the Triazol + RNAeasy plus Kit + TCEP method revealed optimum performance. We successfully achieved isolation of spermatozoal RNA without any spermatozoal DNA contamination from Bos indicus spermatozoa that contains approx. 1000 to 10,000 times less RNA as compared to other mammalian somatic cells. RNA quality was assessed using primers Protamine1 (spermatozoal RNA and spermatozoal DNA), CDH1 (epithelial cell), KIT (germ cell) and PTPRC (leukocytes) designed using primer BLAST where there was no product amplified except Prm1 whose product size was specific for spermatozoal RNA. The results of our investigation on RNA isolation procedures indicate that the inclusion of a disulphide reducing agent (TCEP) is crucial for the process of sperm cell lysis.