{"title":"High-quality total RNA extraction from early-stage lamprey embryos.","authors":"Fumiaki Sugahara, Juan Pascual-Anaya","doi":"10.2144/btn-2023-0004","DOIUrl":null,"url":null,"abstract":"<p><p>High-purity total RNA extraction from animal embryos is essential for transcriptome analyses. lampreys, together with hagfish, are the only extant jawless vertebrates or cyclostomes and are thus key organisms for EvoDevo studies. However, extracting uncontaminated RNA from early-stage embryos remains challenging. RNA does not bind to the silica membrane in filter-based extractions, significantly reducing yields; and ethanol/isopropanol precipitation methods lead to contaminants, bringing down the optical density (OD) 260/280 ratio. The RNA extraction protocol was modified using precentrifugation and adding salts before isopropanol precipitation. This modification significantly increased RNA yield, removed contaminants and improved RNA integrity. Egg membrane sources were suspected to cause RNA purification problems because low-quality extraction does not occur in posthatching embryos.</p>","PeriodicalId":8945,"journal":{"name":"BioTechniques","volume":"74 5","pages":"243-278"},"PeriodicalIF":2.2000,"publicationDate":"2023-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"BioTechniques","FirstCategoryId":"5","ListUrlMain":"https://doi.org/10.2144/btn-2023-0004","RegionNum":4,"RegionCategory":"工程技术","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q3","JCRName":"BIOCHEMICAL RESEARCH METHODS","Score":null,"Total":0}
引用次数: 0
Abstract
High-purity total RNA extraction from animal embryos is essential for transcriptome analyses. lampreys, together with hagfish, are the only extant jawless vertebrates or cyclostomes and are thus key organisms for EvoDevo studies. However, extracting uncontaminated RNA from early-stage embryos remains challenging. RNA does not bind to the silica membrane in filter-based extractions, significantly reducing yields; and ethanol/isopropanol precipitation methods lead to contaminants, bringing down the optical density (OD) 260/280 ratio. The RNA extraction protocol was modified using precentrifugation and adding salts before isopropanol precipitation. This modification significantly increased RNA yield, removed contaminants and improved RNA integrity. Egg membrane sources were suspected to cause RNA purification problems because low-quality extraction does not occur in posthatching embryos.
期刊介绍：
BioTechniques is a peer-reviewed, open-access journal dedicated to publishing original laboratory methods, related technical and software tools, and methods-oriented review articles that are of broad interest to professional life scientists, as well as to scientists from other disciplines (e.g., chemistry, physics, computer science, plant and agricultural science and climate science) interested in life science applications for their technologies.
Since 1983, BioTechniques has been a leading peer-reviewed journal for methods-related research. The journal considers:
Reports describing innovative new methods, platforms and software, substantive modifications to existing methods, or innovative applications of existing methods, techniques & tools to new models or scientific questions
Descriptions of technical tools that facilitate the design or performance of experiments or data analysis, such as software and simple laboratory devices
Surveys of technical approaches related to broad fields of research
Reviews discussing advancements in techniques and methods related to broad fields of research
Letters to the Editor and Expert Opinions highlighting interesting observations or cautionary tales concerning experimental design, methodology or analysis.
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