红豆杉总RNA提取方法的比较

Comparison of Total RNA Extraction Methods from Taxus chinensis

为了确立红豆杉总RNA的最适提取方法,本研究以南方红豆杉嫩叶和曼地亚红豆杉愈伤组织为材料,比较了改良十六烷基三甲基溴化铵(Hexadecyl trimethyl ammonium bromide,CTAB)法、试剂盒法和Trizol法提取总RNA的效果。分别采用紫外分光光度计、凝胶电泳、传统m RNA差异显示技术(differential display of reverse transcriptional PCR,DDRT-PCR)和实时定量PCR(quantitative real-time PCR,qRT-PCR)等技术,测定三种方法提取叶片和细胞系总RNA的浓度、纯度和完整性。结果表明,试剂盒法和改良CTAB法均可以从两种材料中获得较好的提取效果,无RNA降解。相比较而言,CTAB法提取红豆杉愈伤组织和嫩叶总RNA质量最好,浓度较高,OD260/OD280分别为2.44、2.21,OD260/OD230分别为2.11、2.10,浓度分别为621.77 ng/μL、521.10 ng/μL;试剂盒法提取愈伤组织和嫩叶总RNA的OD260/OD280分别为1.98、0.73,OD260/OD230分别为2.15、1.81,浓度分别为609.95 ng/μL、381.53 ng/μL;本研究中Trizol法仅可以从愈伤组织中提取出总RNA,并且提取的总RNA略有降解,伴有少量多糖或蛋白污染,测定的OD260/OD280为1.87,OD260/OD230为2.04,浓度为669.07 ng/μL。经DDRT-PCR和qRT-PCR验证,试剂盒法和改良CTAB法提取的总RNA能够直接用于qRT-PCR等相关实验,并为提取高质量红豆杉总RNA提供借鉴和参考。

In order to establish the optimal extraction method of total RNA from Taxus L.,the leaves of Taxus chinensis var.mairei and callus of Taxus×media were used as material,this study compared the effects of total RNA extraction by modified hexadecyl trimethyl ammonium bromide(CATB)method,the Kit method and the Trizol method.Determination of the concentration,purity and integrity of total RNA from leaves and cell lines by ultraviolet spectrophotometer,gel electrophoresis,differential display of reverse transcriptional PCR(DDRT-PCR),and quantitative real-time PCR(qRT-PCR)technology.The results showed that both the Kit method and the modified CTAB method can obtain better extraction results from two materials without RNA degradation.In comparison,the total RNA of the callus and the young leaves of Taxus chinensis was the best,the concentration was higher,the OD260/OD280 were 2.44,2.21,and the OD260/OD230 were 2.11 and 2.10,respectively,and the concentrations were621.77 ng/μL,521.10 ng/μL;OD260/OD280 of total RNA extracted from callus and young leaves were 1.98 and 0.73,respectively,and OD260/OD230 were 2.15 and 1.81,respectively,and the concentrations were 609.95 ng/μL and381.53 ng/μL,respectively.In this study,the Trizol method can only extract total RNA from callus,and the total RNA extracted is slightly degraded with a small amount of polysaccharide or protein contamination.The measured OD260/OD280 is 1.87 and the OD260/OD230 is 2.04.The concentration was 669.07 ng/μL.DDRT-PCR and qRT-PCR confirmed that the total RNA extracted by Kit method and modified CTAB method can be directly used in q RT-PCR and other related experiments,and provide reference and reference for extracting high quality total RNA from Taxus chinensis.