Extraction of RNA from Fresh, Frozen, and Lyophilized Tuber and Root Tissues

• Published in: Journal of agricultural and food chemistry 2007-03, Vol.55 (5), p.1674-1678
• Main Authors: Kumar, G. N. Mohan, Iyer, Suresh, Knowles, N. Richard
• Format: Article
• Language: English
• Subjects: Biological and medical sciences; Brassica napus - genetics; detergents; extraction; Food engineering; Food industries; Freeze Drying; Freezing; Fundamental and applied biological sciences. Psychology; General aspects; ginger root; Ipomoea batatas - genetics; Plant Roots - genetics; Plant Tubers - genetics; potatoes; radishes; Raphanus - genetics; Rhizome - genetics; RNA; RNA, Plant - isolation & purification; sodium chloride; sodium dodecyl sulfate; sodium sulfite; Solanum tuberosum; Solanum tuberosum - genetics; Starch and starchy product industries; sweet potatoes; turnips; Zingiber officinale - genetics
• ISSN: 0021-8561; 1520-5118
• DOI: 10.1021/jf062941m

A method for isolating trancriptionally 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 μg g fresh weight-1 and 1584 μg g dry weight-1 from frozen and lyophilized samples, respectively. A 260/A 230 ratios of potato RNA extracts were 2.2 or greater, indicating minimal contamination by polyphenols and carbohydrates. Similarly, A 260/A 280 ratios exceeded 1.9, demonstrating minimal contamination of the RNA by tuber protein. While A 260/A 280 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), A 260/A 230 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. Keywords: Solanum tuberosum L.; potato; radish; turnip; ginger; RNA extraction; lyophilized tissue; RT-PCR; starch; polyphenols