从红麻花药和花瓣中提取高质量总RNA的方法

A Method of Extracting High Quality Total RNA from the Anthers and Petals of Kenaf

红麻（Hibiscus cannabinus L.）花药和花瓣因含有较多的多糖、酚类等物质而较难提取高质量的RNA,本研究探索一种适用于红麻花药和花瓣高质量总RNA的方法以进行c DNA文库构建等后续试验。通过借鉴植物DNA的CTAB提取法,结合RNA的理化性质,在提取液中加入6%的β-巯基乙醇,并在开始裂解时加入1/10体积的5 mol·L-1 KAc和1/10体积的无水乙醇对去除多糖特别有效。结果显示,利用该方法得到的RNA质量高,条带清晰完整,检测OD260/280在1.8~2.1之间,OD260/230在2.0~2.6之间,产量为花药987.3 ng·μL-1,花瓣752.1 ng·μL-1。该方法提取的总RNA可直接用于m RNA纯化,c DNA文库构建及RTPCR等后续分子试验。

The anthers and petals of Kenaf contain high content polysaccharide and polyphenol. An efficient method for high quality total RNA extraction from the anthers and petals of Kenaf is requisite for the following experiments such as cDNA library construction and so on. Based on the CTAB extraction method for plant DNA and combined with the physicochemica] properties of RNA. 6% 13- mercapto ethanol was added to extraction buffer and 1/10 volume 5 mol·L-1 KAc with 1/10 volume anhydrous ethanol was used in the initial cell disruption so as to remove polysaccharide from the anthers and petals. The result showed that by this improved method, high quality RNA could be obtained, and the band was clear and complete. A D260/D280 ratio ranged between 1.8 and 2.1. A D260/D230 ratio stand between 2.0 and 2.6. The RNA productivities from the anthers and petals of kenaf were 987.3 ng·μL^-1 and 752.1 ng·μL^-1 respectively. High quality RNA could be obtained by this improved RNA extraction protocol, and RNA was suitable for RT-PCR, mRNA purification, cDNA library construction and so on.