National Peanut Research Laboratory, USDA-ARS, PO Box 509, 1011 Forrester Dr. SE, Dawson, GA 39842, USA.
Mol Biol Rep. 2013 Feb;40(2):1563-8. doi: 10.1007/s11033-012-2204-9. Epub 2012 Oct 29.
Isolation of good quality RNA and DNA from seeds is difficult due to high levels of polysaccharides, polyphenols, and lipids that can degrade or co-precipitate with nucleic acids. Standard RNA extraction methods utilizing guanidinium-phenol-chloroform extraction has not shown to be successful. RNA isolation from plant seeds is a prerequisite for many seed specific gene expression studies and DNA is necessary in marker-assisted selection and other genetic studies. We describe a modified method to isolate both RNA and DNA from the same seed tissue and have been successful with several oil seeds including peanut, soybean, sunflower, canola, and oil radish. An additional LiCl precipitation step was added to isolate both RNA and DNA from the same seed tissues. High quality nucleic acids were observed based on A(260)/A(280) and A(260)/A(230) ratios above 2.0 and distinct bands on gel-electrophoresis. RNA was shown to be suitable for reverse transcriptase polymerase chain reaction based on actin or 60S ribosomal primer amplification and DNA was shown to have a single band on gel-electrophoresis analysis. This result shows that RNA and DNA isolated using this method can be appropriate for molecular studies in peanut and other oil containing seeds.
从种子中分离高质量的 RNA 和 DNA 很困难，因为其中含有高水平的多糖、多酚和脂质，这些物质可能会降解或与核酸共沉淀。利用胍盐-酚-氯仿提取的标准 RNA 提取方法并不成功。从植物种子中分离 RNA 是许多种子特异性基因表达研究的前提条件，而 DNA 在标记辅助选择和其他遗传研究中也是必需的。我们描述了一种改良的方法，可从同一种子组织中同时分离 RNA 和 DNA，并已成功应用于几种油料种子，包括花生、大豆、向日葵、油菜和油萝卜。在同一种子组织中同时分离 RNA 和 DNA 时，增加了一个 LiCl 沉淀步骤。根据 A(260)/A(280) 和 A(260)/A(230) 比值大于 2.0 以及凝胶电泳上的明显条带，可以观察到高质量的核酸。根据肌动蛋白或 60S 核糖体引物扩增，证明 RNA 适合于逆转录聚合酶链反应，并且 DNA 在凝胶电泳分析中显示出单一条带。该结果表明，使用该方法分离的 RNA 和 DNA 可适用于花生和其他含油种子的分子研究。
