一种高效提取棉花不同组织总RNA的热硼酸改良法

A Modified Hot Borate Method for Efficient Isolation of Total RNA from Different Cotton Tissues

针对棉花组织中酚类化合物、多糖、次级代谢产物的含量较多,以及内源RNase活性高等特点,将硼酸缓冲体系、蛋白酶K消化蛋白和氯化锂选择性沉淀RNA步骤偶联在一起,发展成一种有效提取棉花不同组织特别是棉纤维组织总RNA的热硼酸改良法。在提取RNA过程中,不需用酚/氯仿/异戊醇进行抽提,简化了RNA提取的操作步骤。从不同棉花组织提取的RNA质量均能满足cDNA文库的构建、差异显示、cDNA末端的快速扩增(RACE)以及Northern杂交等分子实验。

The isolation of RNA from various tissues of cotton is notoriously difficult due to the abundance of soluble polyphenolic compounds, polysaccharides and second metabolites. Based on the hot borate methods of Hall,et al and Wan,et al, an efficient method coupling extraction in borate buffer with proteinase K digestion protein and selective precipitate of RNA in the presence of LiCl, was developed for the total RNA isolation from cotton (Gossypium hirsutum L.) tissues including roots, stems, leaves, flowers, ovules, seeds and different developing fibers in this paper.The RNA extraction buffer: 200 mmol·L^(-1) Sodium borate decahydrate, pH 9.0, 30 mmol·L^(-1) ethylene glycol bis((-aminoethyl ether )-N,N(-tetraacetic acid (EGTA), 1% sodium dodecyl sulfate (SDS), 1% sodium deoxycholate (DOC), 2% polyvinylpyrrolidone (PVP) (Mr, 40,000), and 10 mmol·L^(-1) dithiothreitol (DTT). The process: (1) The RNA extraction buffer was preheated at 80℃ for 10 min. (2) Grinded 5 g tissue to fine powder with a mortar and a pestle under liquid nitrogen, transferred the frozen powdered tissue into a 50ml precooled polypropylene tube, added 25 ml preheated RNA extraction buffer, and homogenized for 2 min. (3) The homogenate was added with 1ml of proteinase K (20 mg·ml^(-1)), and incubated on a rotary shaker at 100 r·min^(-1) for 1.5 h at 42℃. (4) The homogenate was adjusted to 160 mmol·L^(-1) potassium chloride with 2 mol·L^(-1) KCl, and chilled on ice for 1 h. (5)Centrifugalized the liquid at 12000 r·min^(-1) at 4℃ for 30 min. (6) Transferred the aqueous phase to a new 50ml precooled polypropylene tube, and added 1/4 volume of 10 mol·L^(-1) LiCl. The RNA was precipitated overnight (at least 6 h) at 4℃. (7) Centrifugalized the liquid at 12000 r·min^(-1) for 30 min at 4℃. (8) Decanted the supernatant carefully, washed pellet with 5 ml of precooled 2 mol·L^(-1) LiCl and Centrifugalized the liquid at 12000 r·min^(-1) for 20 min at 4℃. (9) Repeated step (8) three times. (10) The RNA pellet was dissolved in 1600 μl of DEPC treated H2O, transferred to two 1.5 ml microfuge tube and clarified by centrifugation of 12800 r·min^(-1) at 4℃ for 10 min. (11) The supernatant was transferred to a new 1.5 ml microfuge tube, added 1/4 volume of 2 mol·L^(-1) potassium acetate, chilled on ice for 15 min and centrifugalized at 12800 r·min^(-1) for 10 min at 4℃. (12) The supernatant was transferred to a new 1.5 ml microfuge tube, added 2.5 volume of absolute ethanol, mixed by vortexing and placed at -70℃ for 1 to 2 h. (13) The RNA was pelleted by centrifuge of 12800 r·min^(-1) for 30 min at 4℃, washed with 70% cold ethanol, dried briefly and disssoved in DEPC treated H2O. (14) A small sample was taken for spectrophotometric analysis and resolution on 1.2% agrose gel in 1×TBE buffer. The remaining RNA preparation was stored at -80℃.The hot borate RNA procedure utilizes borate, an inhibitor of diphenol oxidase, at alkaline pH to minimize interference by polyphenolics during homogenization. Subsequent treatment by proteinase K inactivates many enzymes, including RNases, and reduces the formation of protein-phenolic complexes that may hinder the recovery of RNA. Following the selective precipitation of RNA by LiCl, inclusion of a salt-precipitation step with potassium acetate, separated the RNA from DNA and removed salt-insoluble materials, including residual polysaccharides and detergents. Thereby, the method can eliminate the interference by high levels of endogenous polysaccharides, phenolics, secondary metabolites during RNA isolation. The procedure was simplified by omitting the extraction steps with supernatant filtration by miracloth and with phenol, chloroform and isoamyl alcohol. The isolated RNA has been proven to be satisfactory for the construction of cDNA (library), DDRT-PCR, RACE PCR and Northern Blotting.