微囊藻群体总RNA提取方法的比较

Comparison of RNA extraction methods from Microcystis colonies

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 Fast RNAPro Blue Kit和方法 4RNeasy 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q PCR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 Fast RNA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 Fast RNA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 q PCR. 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( Fast RNA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.