Increasing evidence of sperm RNA's role in fertilization and embryonic development has provided impetus for its isolation and thorough characterization. Sperm are considered tough-to-lyse cells, due to compact condensed DNA in sperm heads. Lack of consensus among bovine sperm RNA isolation protocols introduces experimental variability in transcriptome studies. Here, we describe an optimized method for total RNA isolation from bovine sperm using TRIzol reagent. This study critically investigated effects of various lysis conditions on sperm RNA isolation. Sperm suspended in TRIzol were subjected to a combination of mechanical treatments (sonication and passage through a 30G needle and syringe) and chemical treatments (supplementation with reducing agents DTT (1,4-dithiothreitol) and TCEP [Tris(2-carboxyethyl) phosphine hydrochloride)]. Microscopic evaluation of sperm lysis confirmed preferential sperm tail versus sperm head lysis. Interestingly, only TCEP-supplemented TRIzol (both mechanical treatments) had progressive sperm head lysis and consistently yielded total-sperm RNA. Furthermore, RNA integrity was confirmed based on electrophoresis profile and an absence of genomic DNA and somatic cells (e.g., epithelial cells, spermatids, etc.) with RT-qPCR. Our findings highlighted the importance of sperm lysis, specifically of sperm head using TCEP with mechanical treatment, in total RNA isolation, and present a bovine-specific sperm RNA isolation method to reduce experimental variabilities.

中文翻译：

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优化牛精子总 RNA 分离，增强精子头部裂解。

越来越多的证据表明精子 RNA 在受精和胚胎发育中的作用，为其分离和彻底表征提供了动力。精子被认为是难以裂解的细胞，因为精子头部的 DNA 紧密浓缩。牛精子 RNA 分离方案之间缺乏共识，导致转录组研究中存在实验变异。在这里，我们描述了一种使用 TRIzol 试剂从牛精子中分离总 RNA 的优化方法。这项研究严格研究了各种裂解条件对精子 RNA 分离的影响。悬浮在 TRIzol 中的精子接受机械处理（超声处理和通过 30G 针头和注射器）和化学处理（补充还原剂 DTT（1,4-二硫苏糖醇）和 TCEP [三（2-羧乙基）膦盐酸盐)]。精子裂解的显微镜评估证实精子尾部优先裂解而不是头部裂解。有趣的是，只有补充 TCEP 的 TRIzol（均为机械处理）具有渐进性精子头部裂解并持续产生总精子 RNA。此外，通过 RT-qPCR，基于电泳图谱以及基因组 DNA 和体细胞（例如，上皮细胞、精子细胞等）的不存在，确认了 RNA 完整性。我们的研究结果强调了精子裂解的重要性，特别是使用机械处理的 TCEP 裂解精子头，在总 RNA 分离中的重要性，并提出了一种牛特异性精子 RNA 分离方法，以减少实验变异。