缺刻缘绿藻总RNA提取方法的比较研究

Comparison of Total RNA Extracted from Myrmecia incisa Using Different Methods

为了获得高质量的缺刻缘绿藻总RNA,采用CTAB法、TRIzol法和RNAplant法3种方法对该藻总RNA的提取效果进行比较分析。结果表明,采用CTAB法提取获得的总RNA产率最高,但因可能含有较多的蛋白质、多糖等杂质使得纯度最低;TRIzol法提取的总RNA纯度较CTAB法稍高,但产率最低,且仍含有DNA带;RNAplant法提取的总RNA电泳图清晰,OD260nm/OD280nm值为2.0,高于前2种方法,产率在2种方法之间,表明RNAplant法更适于缺刻缘绿藻总RNA的提取。对后者提取的总RNA,经反转录合成的cDNA产物大小在500 bp^2 kb,并能通过RT-PCR获得18 SrRNA基因的特异性片段,进一步说明利用RNAplant法可为基因克隆、表达分析等后续研究提供高质量的RNA。

CTAB and TRIzol reagent methods and RNAplant kit method were chosen for a comparison analysis of total RNA extraction from Myrmecia incisa.Total RNA extracted by CTAB method had the highest yield,but the worst quality due to possible contamination of proteins or polysaccharides.The purity of total extracted RNA by TRIzol method was slightly higher than that by CTAB,but the lowest yield（52 μg/100 mg fresh weight of algae）,as well the occurrence of an unexpected DNA band revealed by electrophoresis.By use of the RNAplant kit method,the ratio value of OD260 nm/OD280 nm of total extracted RNA was about 2.0,as well its clear electrophoresis pattern showing the good quality and high purity.The double strand cDNAs synthesized by reverse transcription with the total RNA extracted by RNAplant kit method ranged from 500 bp to 2 kb in size,and a specific fragment of M.incisa 18 S rRNA gene was amplified by RT-PCR with the synthesized cDNA as a template.These results demonstrated that the RNAplant kit method could be suitable for total RNA extraction from this alga and could meet the needs of most molecular biological experiments including gene cloning and expression analysis.