Optimisation of total RNA isolation method from bird sperm

Abstract

The aim of the current study was to determine the optimal RNA extraction in sperm, to support further molecular applications determining the biological roles of sperm in chicken reproduction. Generally, the major issue with RNA isolation is low quantity of RNA is sperm cells. The recent research done on sperm indicates that RNA isolation protocols are specific to the species. RNA extractions from chicken sperm samples (n=47) were respectively performed using TRIzol ® method in combination with PureLink RNA Mini Kit (TRIzol ® Plus RNA Purification System, Invitrogen) and column-based RNA isolation method (PureLink TM RNA Mini Kit, Invitrogen). Both methods were used according to the manufacturer’s protocol but with small modifications. DNase treatment (PureLink ® DNase Set, Invitrogen) was performed in order to eliminate contaminating genomic DNA from samples. To test the quantity and quality the isolated total RNA was checked by the NanoDrop (Thermo Scientific, Wilmington, DE). In general, higher yields were obtained by pure column based isolation when compared to combined TRIzol ® and column based approach. The mean of the yields of total RNA samples ranged from 36.93 ± 2.8 ng (TRIzol ® ) to 381.77 ± 191.3 ng (PureLink TM ). All RNA samples had spectrophotometry values between 1.40 – 1.45 (TRIzol ® ); 1.78 – 1.90 (PureLink TM ) for absorbance ratios A260/A280, and between 0.52 – 1.21 (TRIzol ® ); 1.38 – 2.23 (PureLink TM ) for ratios A260/A230. These results demonstrate that the most suitable protocol of total RNA extraction from bird sperm is column-based RNA isolation method and should be further tested in studies on fertility in birds.