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

Authors


X.-Q. He, State Key Laboratory of Protein and Plant Gene Research, College of Life Sciences, Peking University, Beijing 100871, PR China, and Shou-Jin Fan, Provincial Key Laboratory of Plant Stress, College of Life Sciences, Shandong Normal University, Jinan, Shandong 250014, PR China. E-mail: hexq@pku.edu.cn; fansj@sdnu.edu.cn

ABSTRACT

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 phenol–chloroform and chloroform–isoamyl 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 × g) were carried out at 4°C to avoid degradation.

Results

The procedure took only 1.5 h and was suitable for different cotton tissues. The A260:A280 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 CTAB–ammonium acetate method is easy, rapid, low-cost and effective for high-quality RNA isolation from polysaccharide- and polyphenol-rich cotton tissues. Copyright © 2012 John Wiley & Sons, Ltd.

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