{"title":"Development and optimization of an efficient RNA isolation protocol from bovine (Bos indicus) spermatozoa","authors":"","doi":"10.1016/j.bbrep.2024.101862","DOIUrl":null,"url":null,"abstract":"<div><div>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 <em>Bos indicus</em>. 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 <em>Bos indicus</em> spermatozoa that contains approx. 1000 to 10,000 times less RNA as compared to other mammalian somatic cells. RNA quality was assessed using primers <em>Protamine1</em> (spermatozoal RNA and spermatozoal DNA), <em>CDH1</em> (epithelial cell), <em>KIT</em> (germ cell) and <em>PTPRC</em> (leukocytes) designed using primer BLAST where there was no product amplified except <em>Prm1</em> 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.</div></div>","PeriodicalId":8771,"journal":{"name":"Biochemistry and Biophysics Reports","volume":null,"pages":null},"PeriodicalIF":2.3000,"publicationDate":"2024-11-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Biochemistry and Biophysics Reports","FirstCategoryId":"1085","ListUrlMain":"https://www.sciencedirect.com/science/article/pii/S2405580824002267","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q3","JCRName":"BIOCHEMISTRY & MOLECULAR BIOLOGY","Score":null,"Total":0}
引用次数: 0
Abstract
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.
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.