文献类型：期刊文献

中文题名：适宜当归实时荧光定量分析的总RNA提取方法研究

英文题名：An Appropriate Method of Isolating Total RNA from Angelica sinensis for Quantitative RT-PCR

作者：温随超[1];雒军[1,2];王引权[1,2];荔淑楠[1]

第一作者：温随超

机构：[1]甘肃中医学院;[2]甘肃省高校中(藏)药化学与质量研究省级重点实验室

第一机构：甘肃中医药大学

年份：2014

卷号：30

期号：21

起止页码：235

中文期刊名：中国农学通报

外文期刊名：Chinese Agricultural Science Bulletin

收录：CSTPCD;;CSCD:【CSCD_E2013_2014】；

基金：国家自然科学基金项目"海拔梯度影响当归产量与品质形成的生理生态机制研究(81060327);钾素营养对当归品质形成的影响及生理机理研究(81060327)"

语种：中文

中文关键词：当归;总RNA;提取方法;实时荧光定量分析

外文关键词：Angelica sinensis(Oliv.) Diels;;Total RNA;;Extraction method;;Quantitative RT-PCR

摘要：为了有效消除次生物质、多糖和多酚类物质的严重干扰,获得高质量适宜于实时荧光定量分析的当归总RNA,本研究采用CTAB法、试剂盒法和Trizol法分别提取当归根、茎、叶组织的总RNA,并通过紫外可见光分光光度计、非变性琼脂糖凝胶电泳和反转录荧光定量PCR检测总RNA的纯度、产率、完整性及质量。结果表明:CTAB法提取的当归根、茎、叶组织总RNA纯度较高、完整性较好且叶组织总RNA得率较高;试剂盒法所提的总RNA完整性亦较好,但纯度略差且各组织的得率均较低;Trizol法提取的茎组织总RNA能满足荧光定量PCR要求,但提取的根和叶组织总RNA均出现严重污染,无法满足后续研究要求。因此,CTAB法是一种经济、可靠、适宜当归实时荧光定量分析的总RNA提取方法。本研究结果为今后有效开展当归分子生物学研究奠定了方法学基础,也为其他药用植物总RNA的提取提供了参考。
In order to effectively eliminate the severe disturbances of the secondary substances, polysaccharides and polyphenolics and obtain a high-quality RNA from the tissues of Angelica sinensis for quantitative RT-PCR, three methods of CTAB, Kits and Trizol reagent were used for isolations of total RNA from the roots, stems and leaves of Angelica sinensis, respectively. The purity and yield of the total RNA isolated were detected by a UV/VIS spectrometer, the integrity of total RNA isolated was analyzed by the nondenaturing agarose gel electrophoresis and the quality of total RNA isolated was validated by the quantitative RT-PCR. The results showed that RNAs of the roots, stems and leaves isolated by CTAB method had a better integrity and purity, and the yield of leaves was higher than that of the roots and stems. The total RNAs isolated by Kits method had a good integrity, but there were a lower yield and poorer purity; RNAs of the stems by Trizol reagent could be used for the quantitative RT-PCR, but RNAs of the roots and the leaves were highly polluted, which was ineligible for the subsequent operations. Therefore, CTAB method could be indicated as an economical, reliable and applicable method for isolating total RNA from Angelica sinensis for quantitative RT-PCR. This work established a methodology basis for the research on the molecular biology of Angelica sinensis, and also provided a reference for the total RNA extraction of other medicinal plants.