Comparison of RNA extraction methods from <i>Microcystis</i> colonies

Abstract

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.;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.