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An Improved CTAB-Ammonium Acetate Method for Total RNA Isolation from Cotton

文献类型： 外文期刊

作者： Zhao, Lu 2 ; Ding, Qi 1 ; Zeng, Jun 1 ; Wang, Fu-Rong 3 ; Zhang, Jun 3 ; Fan, Shou-Jin 2 ; He, Xin-Qiang 1 ;

作者机构： 1.Peking Univ, Coll Life Sci, State Key Lab Prot & Plant Gene Res, Beijing 100871, Peoples R China

2.Shandong Normal Univ, Prov Key Lab Plant Stress, Coll Life Sci, Jinan 250014, Shandong, Peoples R China

3.Shandong Cotton Res Ctr, Minist Agr, Key Lab Cotton Breeding & Cultivat Huang Huai Hai, Jinan 250100, Shandong, Peoples R China

关键词： CTAB; RNA isolation; ammonium acetate; cotton

期刊名称：PHYTOCHEMICAL ANALYSIS （ 影响因子：3.373； 五年影响因子：2.959 ）

ISSN： 0958-0344

年卷期： 2012 年 23 卷 6 期

页码：

收录情况： SCI

摘要： Introduction Cotton is an important economic crop. Genetic, developmental and molecular studies of cotton require high-quality total RNA from different tissues. Due to the richness in polyphenols and polysaccharides, the Trizol-based methods and other commercial kits are unsuitable for RNA isolation from cotton. Available methods are generally laborious and time-consuming. Objective To develop an easy, simple and rapid cetyltrimethylammonium bromide (CTAB)ammonium acetate protocol that takes less time and obtains high yield and quality of RNA from polysaccharide- and polyphenol-rich cotton tissues. Methodology Based on the original CTAB protocol, we used phenolchloroform and chloroformisoamyl alcohol to remove proteins, polysaccharides and polyphenols, and ammonium acetate to precipitate RNA, reducing the incubation time prior to RNA precipitation. After adding ammonium acetate to precipitate RNA, all centrifugation steps (14000 x g) were carried out at 4 degrees C to avoid degradation. Results The procedure took only 1.5 h and was suitable for different cotton tissues. The A(260):A(280) ratios ranged from 1.80 to 1.85 with clear 28 s and 18 s ribosomal RNA bands in 1.2% agarose gel. The isolated RNA was usable for downstream molecular studies, such as reverse transcription polymerase chain reaction (PCR) and real-time quantitative PCR. Conclusion The CTABammonium acetate method is easy, rapid, low-cost and effective for high-quality RNA isolation from polysaccharide- and polyphenol-rich cotton tissues. Copyright (c) 2012 John Wiley & Sons, Ltd.

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