title: Spermatozoa input concentrations and RNA isolation methods on RNA yield and quality in bull (Bos taurus).
abstract: Sperm RNA can be used to understand the past spermatogenic process, future successful fertilization, and embryo development. To study the sperm RNA composition and function, isolation of good quality RNA with sufficient quantity is essential. The objective of this study was to assess the influence of sperm input concentrations and RNA isolation methods on RNA yield and quality in bull sperm. The fresh semen samples from bulls (n = 6) were snap-frozen in liquid nitrogen and stored at -80 °C. The sperm RNA was isolated using membrane-based methods combined with TRIzol (RNeasy+TRIzol and PureLink+TRIzol) and conventional methods (TRIzol, Double TRIzol, and RNAzol RT). Based on fluorometric quantification, combined methods resulted in significantly (P < 0.05) higher total RNA yields (800-900 ng/30-40 × 10(6)) as compared with other methods and yielded 20 to 30 fg of RNA/spermatozoon. The quality of RNA isolated by membrane-based methods was superior to that isolated by conventional methods. The sperm RNA was observed to be intact as well as fragmented (50-2000 bp). The study revealed that the membrane-based methods with a cocktail of lysis solution and an optimal input concentration of 30 to 40 million sperm were optimal for maximum recovery of RNA from bull spermatozoa.
authors.affiliation: Reproductive Physiology Laboratory, Animal Physiology Division, ICAR-National Institute of Animal Nutrition and Physiology, Adugodi, Bengaluru 560030, India; Department of Biochemistry, Jain University, Bengaluru 560001, India.
authors.forename: Sivashanmugam
authors.initials: S
authors.lastname: Parthipan
authors.affiliation: Reproductive Physiology Laboratory, Animal Physiology Division, ICAR-National Institute of Animal Nutrition and Physiology, Adugodi, Bengaluru 560030, India. Electronic address: selvarajuars@rediffmail.com.
authors.forename: Sellappan
authors.initials: S
authors.lastname: Selvaraju
authors.affiliation: Reproductive Physiology Laboratory, Animal Physiology Division, ICAR-National Institute of Animal Nutrition and Physiology, Adugodi, Bengaluru 560030, India; Department of Biochemistry, Jain University, Bengaluru 560001, India.
authors.forename: Lakshminarayana
authors.initials: L
authors.lastname: Somashekar
authors.affiliation: Omics Laboratory, Animal Nutrition Division, ICAR-National Institute of Animal Nutrition and Physiology, Adugodi, Bengaluru 560030, India.
authors.forename: Atul P
authors.initials: AP
authors.lastname: Kolte
authors.affiliation: Reproductive Physiology Laboratory, Animal Physiology Division, ICAR-National Institute of Animal Nutrition and Physiology, Adugodi, Bengaluru 560030, India.
authors.forename: Arunachalam
authors.initials: A
authors.lastname: Arangasamy
authors.affiliation: Reproductive Physiology Laboratory, Animal Physiology Division, ICAR-National Institute of Animal Nutrition and Physiology, Adugodi, Bengaluru 560030, India.
authors.forename: Janivara Parameswaraiah
authors.initials: JP
authors.lastname: Ravindra
mesh_terms: D000818:Animals; D002417:Cattle / Q000502:physiology; D015925:Cryopreservation / Q000662:veterinary*; D008297:Male; D012313:RNA / Q000737:chemistry / Q000302:isolation & purification*; D013091:Spermatogenesis; D013094:Spermatozoa / Q000737:chemistry* / Q000166:cytology
chemical_list: D012313:RNA
keywords: Bull; RNA isolation; RNA quality; RNA yield; Spermatozoa