Isolation of High Quality RNA from Cereal Seeds Containing High Levels of Starch
Isolation of High Quality RNA from Cereal Seeds Containing High Levels of Starch
复制标题
从淀粉含量高的谷物种子中分离出高质量 RNA
DOI：
10.1002/pca.1337
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发表时间：
2012-03-01
影响因子：
3.3
通讯作者：
Song, Rentao
中科院分区：
文献类型：
--
作者：
Wang, Guifeng;Wang, Gang;Song, Rentao
谷物是食品、饲料和燃料的重要来源，全球需求迅速增长。然而，谷物种子含有高水平的淀粉和多糖，使高品质的RNA的分离非常困难。目的-开发一种新的方法提取高品质的总RNA从各种淀粉和多糖丰富的谷物种子，如玉米，水稻，高粱和wheat.Methodology -我们开发了一种改进的十二烷基硫酸钠（SDS）/TRIzol方法。在TRIzol提取之前组合使用Tris缓冲液（pH 9.0）和SDS有效地解决了种子匀浆在这种缓冲液中固化的问题。单独使用高浓度的SDS，不仅促进细胞裂解，而且有效地溶解含有高水平淀粉的种子样品。此外，用0.1 M柠檬酸盐缓冲液（pH 4.3）饱和的酸性苯酚用于将RNA与DNA、蛋白质和高水平淀粉分离。这种快速的协议进行了比较与其他RNA提取方法优先用于植物富含多糖和次生代谢产物。结果-凝胶电泳分析表明，提取的总RNA具有良好的完整性，没有明显的DNA污染。此外，约2.0的A（260/280）比、大于2.0的A（260/230）比和大于8.6的RIN值表明分离的RNA具有高纯度。分离的RNA适用于随后的分子操作，例如逆转录聚合酶链反应（PCR）、快速扩增cDNA末端（RACE）和实时PCR。结论-该研究描述了一种简单、高效且重复性高的从各种谷物种子中分离RNA的方法。版权所有（C）2011约翰威利父子有限公司
Introduction - Cereals are an important source of food, feed and fuel with a rapidly increasing global demand. However, cereal seeds contain high levels of starch and polysaccharides, making the isolation of high quality RNA extremely difficult.Objective - To develop a novel method for extracting high quality total RNA from various starch- and polysaccharides-rich cereal seeds, such as maize, rice, sorghum and wheat.Methodology - We developed a modified sodium dodecyl sulphate (SDS)/TRIzol method. The combined use of a Tris buffer (pH 9.0) and SDS before TRIzol extraction effectively resolved the problem of seed homogenate solidification in such a buffer. A high concentration of SDS was used separately, not only to promote cell lysis but also to effectively dissolve seed sample containing high levels of starch. Moreover, acid phenol saturated with 0.1 M citrate buffer (pH 4.3) was used to separate RNA from DNAs, proteins and high levels of starch. This rapid protocol was compared with other RNA isolation methods preferentially used for plants rich in polysaccharides and secondary metabolites.Results - Gel electrophoresis analysis indicated that the extracted total RNA had good integrity without apparent DNA contamination. Furthermore, an A(260/280) ratio of approximately 2.0, an A(260/230) ratio of more than 2.0 and RIN values of more than 8.6 indicated that the isolated RNA was of high purity. The isolated RNA was suitable for subsequent molecular manipulations, such as reverse-transcription polymerase chain reaction (PCR), rapid amplification of cDNA ends (RACE) and real-time PCR.Conclusion-The study has described an easy, efficient and highly reproducible method for RNA isolation from various cereal seeds. Copyright (C) 2011 John Wiley & Sons, Ltd.