A reliable and efficient method for total rna isolation from various members of spurge family (Euphorbiaceae).
A reliable and efficient method for total rna isolation from various members of spurge family (Euphorbiaceae).
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DOI：
10.1002/pca.1205
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发表时间：
2010-09
期刊：
影响因子：
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通讯作者：
Jia Xu;Mahender Aileni;S. Abbagani;Peng Zhang
中科院分区：
文献类型：
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作者：
Jia Xu;Mahender Aileni;S. Abbagani;Peng Zhang
为了对任何一个科的植物进行分子生物学研究，提取高质量和完整的RNA是前提和关键。大戟科成员以其高水平的蜡、多糖油、多酚和次级代谢产物而闻名。这些条件被认为不利地干扰RNA分离的各种方法。目的建立一种简便、快速、重复性好的十六烷基三甲基溴化铵（CTAB）提取方法，在不降低质量和产量的前提下，缩短从橡胶树、蓖麻、麻风树、木薯等木本灌木叶片和块根组织中提取RNA的时间和成本。esculenta）。方法简单的修改和快速的步骤，介绍了原来的CTAB协议。所有离心步骤均在4°C下以12000 rpm进行10 min，减少样品重量并省略亚精胺和LiCl的使用，从而减少RNA沉淀前的孵育时间。这种快速的CTAB协议进行了比较，与各种RNA分离方法用于植物富含多糖和次生代谢产物。结果该方法可在2 h内完成，并可同时处理多个样品。高质量的RNA可以从我们尝试过的物种的所有组织中分离出来。从不同物种中分离的RNA用作RT-PCR分析的稳健模板。结论改进的CTAB法可以快速提取大戟科植物的高质量RNA。
INTRODUCTION 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. OBJECTIVE 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). METHODOLOGY 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. RESULTS 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. CONCLUSION 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.