Parthipan Sivashanmugam, Selvaraju Sellappan, Somashekar Lakshminarayana, Kolte Atul P, Arangasamy Arunachalam, Ravindra Janivara Parameswaraiah
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.
Reproductive Physiology Laboratory, Animal Physiology Division, ICAR-National Institute of Animal Nutrition and Physiology, Adugodi, Bengaluru 560030, India.
Anal Biochem. 2015 Aug 1;482:32-9. doi: 10.1016/j.ab.2015.03.022. Epub 2015 Mar 28.
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.
精子RNA可用于了解过去的精子发生过程、未来的成功受精以及胚胎发育。为了研究精子RNA的组成和功能，分离出高质量且足够数量的RNA至关重要。本研究的目的是评估精子输入浓度和RNA分离方法对公牛精子RNA产量和质量的影响。从公牛（n = 6）采集的新鲜精液样本在液氮中速冻，并储存在-80°C。使用基于膜的方法结合TRIzol（RNeasy+TRIzol和PureLink+TRIzol）以及传统方法（TRIzol、Double TRIzol和RNAzol RT）分离精子RNA。基于荧光定量分析，与其他方法相比，结合方法产生的总RNA产量显著更高（P < 0.05）（800 - 900 ng/30 - 40×10⁶），每个精子产生20至30 fg的RNA。基于膜的方法分离的RNA质量优于传统方法。观察到精子RNA既有完整的，也有片段化的（50 - 2000 bp）。该研究表明，使用裂解液混合物且最佳输入浓度为3000万至4000万个精子的基于膜的方法，对于从公牛精子中最大程度回收RNA是最佳的。
