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引用本文:高胜玲,黄亚新,卢亚萍,施丽梅,孔繁翔,张小倩.微囊藻群体总RNA提取方法的比较.湖泊科学,2018,30(2):441-448. DOI:10.18307/2018.0215
GAO Shengling,HUANG Yaxin,LU Yaping,SHI Limei,KONG Fanxiang,ZHANG Xiaoqian.Comparison of RNA extraction methods from Microcystis colonies. J. Lake Sci.2018,30(2):441-448. DOI:10.18307/2018.0215
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微囊藻群体总RNA提取方法的比较
高胜玲1,2, 黄亚新1, 卢亚萍1, 施丽梅2, 孔繁翔2, 张小倩1
1.南京农业大学生命科学学院, 南京 210095;2.中国科学院南京地理与湖泊研究所湖泊与环境国家重点实验室, 南京 210008
摘要:
微囊藻群体富含多糖类物质是影响微囊藻RNA提取的关键因素.为了获得高质量的微囊藻群体总RNA,对比分析4种针对多糖含量较高的方法——方法1 PGTX-bead法、方法2 CTAB-bead法、方法3 FastRNA® Pro Blue Kit和方法4 RNeasy Mini Kit对微囊藻群体总RNA的提取效果.采用琼脂糖凝胶电泳检测微囊藻群体RNA的完整性,Nanodrop ND1000分光光度计检测RNA纯度及浓度,并采用qPCR检测DNA污染情况.结果表明,4种方法都能从微囊藻群体中提取获得RNA,并在去除DNA后都可以进行RT-PCR等后续实验.方法1 PGTX-bead提取的RNA产量最高,纯度好,DNA污染小,成本低,适合从微囊藻群体中大量提取RNA;方法2 CTAB-bead提取的RNA样品产量也较高,但DNA污染严重,适合需要同时提取DNA和RNA的样本;方法3 FastRNA® Pro Blue Kit和方法4 RNeasy Mini Kit提取的RNA产量都较低,但方法4操作简单,耗时短,所检测目的基因的相对表达量较高,更适合从少量的微囊藻群体中提取总RNA.
关键词:  微囊藻群体  RNA提取  qPCR  多糖
DOI:10.18307/2018.0215
分类号:
基金项目:国家自然科学基金项目(31370509)和江苏省自然科学基金项目(BK20131466)联合资助.
Comparison of RNA extraction methods from Microcystis colonies
GAO Shengling1,2, HUANG Yaxin1, LU Yaping1, SHI Limei2, KONG Fanxiang2, ZHANG Xiaoqian1
1.College of Life Science, Nanjing Agricultural University, Nanjing 210095, P. R. China;2.State Key Laboratory of Lake Science and Environment, Nanjing Institute of Geography and Limnology, Chinese Academy of Sciences, Nanjing 210008, P. R. China
Abstract:
Microcystis colonies are rich in polysaccharide materials which are the key factors influencing RNA extraction. In order to obtain high-quality total RNA from Microcystis colonies, comparative study of several methods including Method 1 PGTX-bead, Method 2 CTAB-bead, Method 3 FastRNA® Pro Blue Kit and Method 4 RNeasy Mini Kit was conducted. All these methods were suitable for polysaccharide-rich materials. The integrity of RNA was analyzed by agarose gel electrophoresis, RNA concentration and purity were detected by Nanodrop ND1000 spectrophotometer, and DNA contamination was determined by qPCR. The results demonstrated that all the four methods were able to extract RNA from Microcystis colonies, and they were able to be used for downstream experiments, such as RT-PCR, after the contaminated DNA was removed. Method 1(PGTX-bead extraction) yielded RNA of highest concentration, good purity, low DNA contamination and it was low cost; Method 2 (CTAB-bead extraction) also yielded RNA of high concentration, but with heavy DNA contamination. This method was suitable for samples that need simultaneous isolation of RNA and DNA. Method 3 (FastRNA® Pro Blue Kit)and Method 4 (RNeasy Mini Kit) extraction yielded RNA of low concentration, but Method 4 had simpler protocol, less time consuming, higher relative expression of the detected function gene, thus was suitable for extracting total RNA from small amount of Microcystis colonies.
Key words:  Microcystis colonies  RNA extraction  qPCR  polysaccharide
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