以羧酸为RNA变性剂的毛细管内变性凝胶电泳高效分离RNA

Keiko Sumitomo, Y. Yamaguchi
{"title":"以羧酸为RNA变性剂的毛细管内变性凝胶电泳高效分离RNA","authors":"Keiko Sumitomo, Y. Yamaguchi","doi":"10.2198/SBK.52.133","DOIUrl":null,"url":null,"abstract":"For RNA size separation in a small sample volume (<10 nL), a strong denaturant to cleave the intramolecular hydrogen bonds that maintain the high-order structures of RNA and optimization for a small sample volume are required. We suggested, “in-capillary denaturing gel electrophoresis” as the RNA separation based on capillary gel electrophoresis, that realizes the denaturation and separation simultaneously in a capillary tube. We found that carboxylic acids were strong denaturants for in-capillary denaturing gel electrophoresis, and the performance of RNA separation was dramatically improved with a running buffer containing acetic acid. Based on the decrease of DNA melting temperature, we estimated that the denaturing ability of 2.0 M acetic acid was stronger than that of either 2.5 M formaldehyde or 7.0 M urea. The baseline separation of RNA with a size of 100−10,000 nt was achieved in only 25 min by in-capillary denaturing gel electrophoresis containing 2.0 M acetic acid. The resolution and number of plates of RNA separation were higher and larger than those obtained in a conventional capillary gel electrophoresis with sample preparation with 7.0 M urea.","PeriodicalId":15059,"journal":{"name":"Journal of capillary electrophoresis","volume":"60 1","pages":"133-138"},"PeriodicalIF":0.0000,"publicationDate":"2008-09-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"1","resultStr":"{\"title\":\"High performance RNA separation by in-capillary denaturing gel electrophoresis with carboxylic acid as RNA denaturant\",\"authors\":\"Keiko Sumitomo, Y. Yamaguchi\",\"doi\":\"10.2198/SBK.52.133\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"For RNA size separation in a small sample volume (<10 nL), a strong denaturant to cleave the intramolecular hydrogen bonds that maintain the high-order structures of RNA and optimization for a small sample volume are required. We suggested, “in-capillary denaturing gel electrophoresis” as the RNA separation based on capillary gel electrophoresis, that realizes the denaturation and separation simultaneously in a capillary tube. We found that carboxylic acids were strong denaturants for in-capillary denaturing gel electrophoresis, and the performance of RNA separation was dramatically improved with a running buffer containing acetic acid. Based on the decrease of DNA melting temperature, we estimated that the denaturing ability of 2.0 M acetic acid was stronger than that of either 2.5 M formaldehyde or 7.0 M urea. The baseline separation of RNA with a size of 100−10,000 nt was achieved in only 25 min by in-capillary denaturing gel electrophoresis containing 2.0 M acetic acid. The resolution and number of plates of RNA separation were higher and larger than those obtained in a conventional capillary gel electrophoresis with sample preparation with 7.0 M urea.\",\"PeriodicalId\":15059,\"journal\":{\"name\":\"Journal of capillary electrophoresis\",\"volume\":\"60 1\",\"pages\":\"133-138\"},\"PeriodicalIF\":0.0000,\"publicationDate\":\"2008-09-15\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"1\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Journal of capillary electrophoresis\",\"FirstCategoryId\":\"1085\",\"ListUrlMain\":\"https://doi.org/10.2198/SBK.52.133\",\"RegionNum\":0,\"RegionCategory\":null,\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"\",\"JCRName\":\"\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Journal of capillary electrophoresis","FirstCategoryId":"1085","ListUrlMain":"https://doi.org/10.2198/SBK.52.133","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"","JCRName":"","Score":null,"Total":0}
引用次数: 1

摘要

对于小样本量(<10 nL)的RNA大小分离,需要一种强变性剂来切割维持RNA高阶结构的分子内氢键,并对小样本量进行优化。我们建议将“毛细管内变性凝胶电泳”作为基于毛细管凝胶电泳的RNA分离方法,在毛细管内同时实现变性和分离。我们发现羧酸是毛细管内变性凝胶电泳的强变性剂,并且含有乙酸的运行缓冲液显著提高了RNA分离的性能。根据DNA熔融温度的降低,我们估计2.0 M乙酸的变性能力比2.5 M甲醛和7.0 M尿素的变性能力强。采用含有2.0 M乙酸的毛细管内变性凝胶电泳,在25分钟内即可实现100 ~ 10,000 nt RNA的基线分离。与常规毛细管凝胶电泳相比,用7.0 M尿素制备样品的RNA分离分辨率更高,分离板数也更大。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
查看原文
分享 分享
微信好友 朋友圈 QQ好友 复制链接
本刊更多论文
High performance RNA separation by in-capillary denaturing gel electrophoresis with carboxylic acid as RNA denaturant
For RNA size separation in a small sample volume (<10 nL), a strong denaturant to cleave the intramolecular hydrogen bonds that maintain the high-order structures of RNA and optimization for a small sample volume are required. We suggested, “in-capillary denaturing gel electrophoresis” as the RNA separation based on capillary gel electrophoresis, that realizes the denaturation and separation simultaneously in a capillary tube. We found that carboxylic acids were strong denaturants for in-capillary denaturing gel electrophoresis, and the performance of RNA separation was dramatically improved with a running buffer containing acetic acid. Based on the decrease of DNA melting temperature, we estimated that the denaturing ability of 2.0 M acetic acid was stronger than that of either 2.5 M formaldehyde or 7.0 M urea. The baseline separation of RNA with a size of 100−10,000 nt was achieved in only 25 min by in-capillary denaturing gel electrophoresis containing 2.0 M acetic acid. The resolution and number of plates of RNA separation were higher and larger than those obtained in a conventional capillary gel electrophoresis with sample preparation with 7.0 M urea.
求助全文
通过发布文献求助,成功后即可免费获取论文全文。 去求助
来源期刊
自引率
0.00%
发文量
0
期刊最新文献
Electrophoretic extraction of protein complexes after separation and detection by a combined method of non-denaturing two-dimensional electrophoresis and reversible staining Use of Escherichia coli expression system for analyzing kinase motifs Proteomic analysis of spheroids of rhabdomyosarcoma cells cultured with decellularized muscle extracts Drug screening and kinase activity profiling of a novel patient-derived cell line of clear cell ovarian carcinoma Proteogenomic approach to drug targets in osteosarcomas with different original sites
×
引用
GB/T 7714-2015
复制
MLA
复制
APA
复制
导出至
BibTeX EndNote RefMan NoteFirst NoteExpress
×
×
提示
您的信息不完整,为了账户安全,请先补充。
现在去补充
×
提示
您因"违规操作"
具体请查看互助需知
我知道了
×
提示
现在去查看 取消
×
提示
确定
0
微信
客服QQ
Book学术公众号 扫码关注我们
反馈
×
意见反馈
请填写您的意见或建议
请填写您的手机或邮箱
已复制链接
已复制链接
快去分享给好友吧!
我知道了
×
扫码分享
扫码分享
Book学术官方微信
Book学术文献互助
Book学术文献互助群
群 号:481959085
Book学术
文献互助 智能选刊 最新文献 互助须知 联系我们:info@booksci.cn
Book学术提供免费学术资源搜索服务,方便国内外学者检索中英文文献。致力于提供最便捷和优质的服务体验。
Copyright © 2023 Book学术 All rights reserved.
ghs 京公网安备 11010802042870号 京ICP备2023020795号-1