Kumar G N Mohan, Iyer Suresh, Knowles N Richard
Postharvest Physiology and Biochemistry Lab, Department of Horticulture and Landscape Architecture, P.O. Box 646414, Washington State University, Pullman, Washington 99164-6414, USA.
J Agric Food Chem. 2007 Mar 7;55(5):1674-8. doi: 10.1021/jf062941m. Epub 2007 Feb 9.
A method for isolating transcriptionally competent RNA from fresh, frozen, and lyophilized plant storage tissues containing high levels of starch and phenolics is described. The protocol avoids the use of guanidium salts, which often lead to the formation of a viscous gel during extraction of high starch-containing tissues, and instead uses a borate-Tris buffer in combination with high concentrations of NaCl, Na2SO3, and sodium dodecyl sulfate in the extraction medium. RNA was extracted from fresh, frozen, and lyophilized tissues of potato tubers, storage roots of sweet potato, radish, and turnip, and rhizomes of ginger. The yield of RNA from potato tubers averaged 281 microg g fresh weight(-1) and 1584 microg g dry weight(-1) from frozen and lyophilized samples, respectively. A260/A230 ratios of potato RNA extracts were 2.2 or greater, indicating minimal contamination by polyphenols and carbohydrates. Similarly, A260/A280 ratios exceeded 1.9, demonstrating minimal contamination of the RNA by tuber protein. While A260/A280 ratios of extracts from the other plant species were somewhat lower than those for potato (average = 1.56 and 1.80 for fresh and lyophilized samples, respectively), A260/A230 ratios averaged more than 2.0, and the RNA extracted from fresh and lyophilized samples of all species was intact, as demonstrated by denaturing agarose-formaldehyde gel electrophoresis. The protocol yielded RNA suitable for downstream molecular applications involving reverse transcription-polymerase chain reaction from all five species. Transcriptionally competent RNA was also recovered from lyophilized potato tuber tissue stored for 6 years (ambient temperature) by a simple modification to the protocol involving extraction in cold acetone. Lyophilization can thus be used to preserve RNA in high starch- and phenolic-containing plant tissues for studies on gene expression.
本文描述了一种从富含淀粉和酚类物质的新鲜、冷冻及冻干植物储存组织中分离具有转录活性RNA的方法。该方案避免使用胍盐，因为在提取高淀粉含量组织时，胍盐常常导致形成粘性凝胶，而是在提取介质中使用硼酸盐 - Tris缓冲液，并结合高浓度的NaCl、Na2SO3和十二烷基硫酸钠。RNA从马铃薯块茎的新鲜、冷冻及冻干组织、甘薯的储存根、萝卜、芜菁以及姜的根茎中提取。马铃薯块茎RNA的产量，冷冻和冻干样品分别平均为281微克/克鲜重(-1)和1584微克/克干重(-1)。马铃薯RNA提取物的A260/A230比值为2.2或更高，表明多酚和碳水化合物污染最小。同样，A260/A280比值超过1.9，表明RNA受块茎蛋白污染最小。虽然其他植物物种提取物的A260/A280比值略低于马铃薯（新鲜和冻干样品分别平均为1.56和1.80），但A260/A230比值平均超过2.0，并且通过变性琼脂糖 - 甲醛凝胶电泳证明，从所有物种的新鲜和冻干样品中提取的RNA都是完整的。该方案产生的RNA适用于所有五个物种的涉及逆转录 - 聚合酶链反应的下游分子应用。通过对方案进行简单修改，即在冷丙酮中提取，还从储存6年（室温）的冻干马铃薯块茎组织中回收了具有转录活性的RNA。因此，冻干可用于在富含淀粉和酚类的植物组织中保存RNA，用于基因表达研究。
