An improved method for RNA isolation and cDNA library construction from immature seeds of Jatropha curcas L.
An improved method for RNA isolation and cDNA library construction from immature seeds of Jatropha curcas L.
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DOI：
10.1186/1756-0500-3-126
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发表时间：
2010-05-05
影响因子：
1.8
通讯作者：
Yin Z
中科院分区：
文献类型：
--
作者：
Sangha JS;Gu K;Kaur J;Yin Z
RNA的质量和数量有时不适合从富含油脂、多糖和其他次生代谢物的植物种子中构建cDNA文库。麻疯树种子的研究(英文)富含脂肪酸/脂类、贮藏蛋白、多糖和许多其他次生代谢物，可与RNA结合和/或共沉淀，使其不适合下游应用。现有的RNA提取方法和商业试剂盒往往不能从未成熟的麻疯树种子中提取高质量的总RNA，用于Poly(A)+RNA的纯化和DNA合成。开发了一种从未成熟麻疯树种子中提取高质量总RNA的方法，其中结合使用了基于CTAB的RNA提取方法和商业植物RNA提取试剂盒的硅胶柱。提取时间由2d缩短至3h左右，可用于Poly(A)+RNA的纯化、cDNA合成、文库构建、RT-PCR和Northern杂交。根据所选克隆的序列信息和扩增的PCR产物，该文库似乎是麻疯树全长基因的良好来源。该方法同样适用于从芥菜和水稻种子中提取RNA。这是一种简单的CTAB+硅胶柱方法，可以从富含油分的未成熟麻疯树种子中提取高质量的RNA，适用于几种下游应用。这种方法提取RNA所需时间较短，对于其他组织也同样有效，在这些组织中，RNA的质量和数量受到脂肪酸、多糖和多酚的高度干扰。
RNA quality and quantity is sometimes unsuitable for cDNA library construction, from plant seeds rich in oil, polysaccharides and other secondary metabolites. Seeds of jatropha (Jatropha curcas L.) are rich in fatty acids/lipids, storage proteins, polysaccharides, and a number of other secondary metabolites that could either bind and/or co-precipitate with RNA, making it unsuitable for downstream applications. Existing RNA isolation methods and commercial kits often fail to deliver high-quality total RNA from immature jatropha seeds for poly(A)+ RNA purification and cDNA synthesis. A protocol has been developed for isolating good quality total RNA from immature jatropha seeds, whereby a combination of the CTAB based RNA extraction method and a silica column of a commercial plant RNA extraction kit is used. The extraction time was reduced from two days to about 3 hours and the RNA was suitable for poly(A)+ RNA purification, cDNA synthesis, cDNA library construction, RT-PCR, and Northern hybridization. Based on sequence information from selected clones and amplified PCR product, the cDNA library seems to be a good source of full-length jatropha genes. The method was equally effective for isolating RNA from mustard and rice seeds. This is a simple CTAB + silica column method to extract high quality RNA from oil rich immature jatropha seeds that is suitable for several downstream applications. This method takes less time for RNA extraction and is equally effective for other tissues where the quality and quantity of RNA is highly interfered by the presence of fatty acids, polysaccharides and polyphenols.