Development of a new method for sperm RNA purification in the chicken

Currently RNA transcripts are being used as male fertility biomarker for many mammalian species, but research work on chicken is at halt because classical RNA isolation methods are not effective for chicken spermatozoa. Hence, attempts have been made to optimize RNA isolation protocol from chicken sperm by using different methods, and to confirm the presence of sperm-specific transcripts of PRM and PLCZ1. Semen samples were centrifuged at low speed for removing debris like uric acid. Further, 1mL diluted semen was gently placed over 40% PureSperm or 45%/90% Percoll, and centrifuged to remove somatic cells and immature diploid spermatocytes. RNA was isolated from sperm by using RNAzol or TRIzol reagent or RNeasy Micro kit with certain modification, and RNA quantity and quality were evaluated. RNA isolated by using RNAzol or RNeasy Micro Kit yielded good quantity and quality of RNA for downstream applications compared to TRIzol. 40% PureSperm was found effective in removing somatic cells. RT-PCR results showed that sperm RNA samples were negative for CD4 and PTPRC. All the sperm RNA samples were positive for PRM and PLCZ1, markers of sperm RNA.