An improved CTAB-ammonium acetate method for total RNA isolation from cotton.
An improved CTAB-ammonium acetate method for total RNA isolation from cotton.
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DOI：
10.1002/pca.2368
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发表时间：
2012-11
期刊：
影响因子：
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通讯作者：
Lu Zhao;Q. Ding;Jun Zeng;Furong Wang;Jun Zhang;S. Fan;Xin He
中科院分区：
文献类型：
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作者：
Lu Zhao;Q. Ding;Jun Zeng;Furong Wang;Jun Zhang;S. Fan;Xin He
棉花是一种重要的经济作物。棉花的遗传、发育和分子生物学研究需要高质量的不同组织的总RNA。由于多酚和多糖的丰富性，基于Trizol的方法和其他商业试剂盒不适合从棉花中分离RNA。现有的方法一般都是费力费时的。目的建立一种简便、快速、快速的从富含多糖和多酚的棉花组织中提取RNA的方法。方法：在原有的CTAB法的基础上，采用苯酚-氯仿和氯仿-异戊醇去除蛋白质、多糖和多酚，乙酸铵沉淀RNA，缩短了RNA沉淀前的孵育时间。加入乙酸铵沉淀RNA后，所有离心步骤（14000 × g）均在4 ℃下进行，以避免降解。结果该方法仅需1.5h，适用于不同棉花组织。A（260）：A（280）比值在1.80 ~ 1.85之间，在1.2%琼脂糖凝胶上有清晰的28 s和18 s核糖体RNA条带。分离的RNA可用于下游分子研究，如逆转录聚合酶链反应（PCR）和实时定量PCR。结论CTAB-醋酸铵法是一种简便、快速、低成本、有效的从富含多糖和多酚的棉花组织中提取高质量RNA的方法。
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 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 CTAB-ammonium acetate method is easy, rapid, low-cost and effective for high-quality RNA isolation from polysaccharide- and polyphenol-rich cotton tissues.