National Key Laboratory of Plant Molecular Genetics, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 300 Fenglin Road, Shanghai 200032, China.
Phytochem Anal. 2010 Sep-Oct;21(5):395-8. doi: 10.1002/pca.1205.
It is prerequisite and crucial to extract RNA with high quality and integrity in order to carry out molecular biology studies in any plant species of a family. Euphorbiaceae members are known for high levels of their waxes, oils with polysaccharides, polyphenolics and secondary metabolites. These conditions are recognised to interfere unfavourably with various methodologies of RNA isolation.
To develop a simple, rapid and reproducible cetyltrimethylamonium bromide (CTAB)-based protocol, to reduce the time and cost of extraction without reducing quality and yield of RNA extracted from various recalcitrant Euphorbiaceae member plant tissues such as from tree leaves (Hevea brasilensis), woody shrubs leaves (Ricinus communis, Jatropha curcas, Manihot esculenta) and storage root tissue (M. esculenta).
Simple modifications and fast steps were introduced to the original CTAB protocol. All centrifugation steps were carried out at 4°C at 12000 rpm for 10 min, the sample weight was decreased and usage of spermidine and LiCl was omitted, reducing incubation time prior to RNA precipitation. This rapid CTAB protocol was compared with various RNA isolation methods intended for use with plants rich in polysaccharides and secondary metabolites.
The procedure can be completed within 2 h and many samples can be processed at the same time. RNA of high quality could be isolated from all the tissues of species that we tried. The isolated RNA from different species served as a robust template for RT-PCR analysis.
The study has shown that the improvement of a CTAB-based protocol allows the rapid isolation of high-quality RNA from various recalcitrant Euphorbiaceae members.
在任何植物科中进行分子生物学研究，提取高质量和完整性的 RNA 是前提和关键。大戟科植物以高水平的蜡质、多糖油、多酚和次生代谢物而闻名。这些条件被认为会对各种 RNA 分离方法产生不利影响。
开发一种简单、快速和可重复的十六烷基三甲基溴化铵（CTAB）- 基于协议，以减少提取时间和成本，而不会降低从各种顽固的大戟科成员植物组织中提取的 RNA 的质量和产量，例如树叶（巴西橡胶树）、木本灌木叶（蓖麻、麻疯树、木薯）和贮藏根组织（木薯）。
对原始 CTAB 方案进行了简单的修改和快速步骤。所有离心步骤均在 4°C 下以 12000 rpm 进行 10 分钟，减少了样品重量，并省略了亚精胺和 LiCl 的使用，减少了 RNA 沉淀前的孵育时间。该快速 CTAB 方案与各种旨在用于富含多糖和次生代谢物的植物的 RNA 分离方法进行了比较。
该程序可在 2 小时内完成，并且可以同时处理多个样品。可以从我们尝试的所有物种的组织中分离出高质量的 RNA。来自不同物种的分离 RNA 可作为 RT-PCR 分析的稳健模板。
研究表明，CTAB 基方案的改进允许从各种顽固的大戟科成员中快速分离高质量的 RNA。
