开发和优化从牛(Bos indicus)精子中分离 RNA 的高效方案

IF 2.3 Q3 BIOCHEMISTRY & MOLECULAR BIOLOGY
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引用次数: 0

摘要

从精子细胞中提取最佳的 RNA 对于进行有效的高通量分析至关重要,这些分析涉及精子在豚鼠生育和其他繁殖过程中的作用。然而,精液由精子和来自雄性生殖系统的其他一些分泌物组成,其中也包括各种体细胞类型。因此,去除体细胞可保证精子 RNA 的纯度。在本研究中,我们测试了五种不同的 RNA 分离方案,并使用分光光度计和聚合酶链反应评估了它们的产量和纯度。在五种 RNA 分离方案中,Triazol + RNAeasy plus Kit + TCEP 方法的性能最佳。与其他哺乳动物体细胞相比,Bos indicus 精子中的 RNA 含量约低 1000 到 10,000 倍,我们成功地从这些精子中分离出了精子 RNA,且没有精子 DNA 污染。使用引物 BLAST 设计的引物 Protamine1(精子 RNA 和精子 DNA)、CDH1(上皮细胞)、KIT(生殖细胞)和 PTPRC(白细胞)对 RNA 质量进行了评估。我们对 RNA 分离程序的研究结果表明,加入二硫化物还原剂(TCEP)对精子细胞裂解过程至关重要。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Development and optimization of an efficient RNA isolation protocol from bovine (Bos indicus) spermatozoa
Achieving the optimum extraction of RNA from spermatozoal cells is crucial for carrying out effective high-throughput analysis regarding its role in fertility and other reproduction processes in Bos indicus. Nevertheless, semen comprises spermatozoa and several other secretions from the male reproductive system, which as well consist of diverse somatic cell types. Therefore, the elimination of somatic cells guarantees the purity of the sperm RNA. In the present study, we tested five different RNA isolation protocols and evaluated them for their yield and purity using spectrophotometer and polymerase chain reaction. Among the five RNA isolation protocols, the Triazol + RNAeasy plus Kit + TCEP method revealed optimum performance. We successfully achieved isolation of spermatozoal RNA without any spermatozoal DNA contamination from Bos indicus spermatozoa that contains approx. 1000 to 10,000 times less RNA as compared to other mammalian somatic cells. RNA quality was assessed using primers Protamine1 (spermatozoal RNA and spermatozoal DNA), CDH1 (epithelial cell), KIT (germ cell) and PTPRC (leukocytes) designed using primer BLAST where there was no product amplified except Prm1 whose product size was specific for spermatozoal RNA. The results of our investigation on RNA isolation procedures indicate that the inclusion of a disulphide reducing agent (TCEP) is crucial for the process of sperm cell lysis.
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来源期刊
Biochemistry and Biophysics Reports
Biochemistry and Biophysics Reports Biochemistry, Genetics and Molecular Biology-Biophysics
CiteScore
4.60
自引率
0.00%
发文量
191
审稿时长
59 days
期刊介绍: Open access, online only, peer-reviewed international journal in the Life Sciences, established in 2014 Biochemistry and Biophysics Reports (BB Reports) publishes original research in all aspects of Biochemistry, Biophysics and related areas like Molecular and Cell Biology. BB Reports welcomes solid though more preliminary, descriptive and small scale results if they have the potential to stimulate and/or contribute to future research, leading to new insights or hypothesis. Primary criteria for acceptance is that the work is original, scientifically and technically sound and provides valuable knowledge to life sciences research. We strongly believe all results deserve to be published and documented for the advancement of science. BB Reports specifically appreciates receiving reports on: Negative results, Replication studies, Reanalysis of previous datasets.
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