Modified methods for bovine sperm RNA isolation for consistent quality and RNA yield

Sperm mRNA transcriptional profiling can be used to evaluate the fertility of breeding bulls. This study aimed to compare the modified RNA isolation methods for higher RNA yield and quality from freshly ejaculated sperm of cattle and buffalo bull for further transcriptome analysis. Ten fresh ejaculates from each Sahiwal (n = 10 bulls x 10 ejaculates) and Murrah bulls (n = 10 bulls x 10 ejaculates) were used for RNA isolation. Swim-up technique was used for live sperm separation and recovery. From the recovered live sperm, total sperm RNA was isolated by conventional methods (TRIzol, Double TRIzol), membrane-based methods combined with TRIzol (RNeasy + TRIzol) with the addition of β-mercaptoethanol (BME) and Kit (RNeasy mini) methods in fresh semen. Among different isolation methods; the membrane-based modified methods combined with TRIzol (RNeasy + TRIzol) with the addition of β-mercaptoethanol (BME) resulted significantly (P < 0.05) higher total RNA quantity (300-340 ng/μL) and better purity in different concentrations of spermatozoa viz., 30-40 million, 70-80 million and 300-400 million sperm. The study concluded that the inclusion of BME to the combined membrane-based methods with somatic cell lysis buffer solution was best for constant increased yield and purity of RNA isolation from Sahiwal cattle and Murrah buffalo bull sperm. This method will help with the interpretation of data from animal models and the consistency of clinical assessments of male factor fertility employing RNA molecular biomarkers.