Extraction of RNA from fresh, frozen, and lyophilized tuber and root tissues.
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.
Kumar
G N Mohan
GN
Postharvest Physiology and Biochemistry Lab, Department of Horticulture and Landscape Architecture, P.O. Box 646414, Washington State University, Pullman, Washington 99164-6414, USA.
Iyer
Suresh
S
Knowles
N Richard
NR
D029688:Brassica napus / Q000235:genetics; D005612:Freeze Drying; D005615:Freezing; D020939:Zingiber officinale / Q000235:genetics; D027723:Ipomoea batatas / Q000235:genetics; D018517:Plant Roots / Q000235:genetics*; D035281:Plant Tubers / Q000235:genetics*; D018749:RNA, Plant / Q000302:isolation & purification*; D031224:Raphanus / Q000235:genetics; D027343:Rhizome / Q000235:genetics; D011198:Solanum tuberosum / Q000235:genetics
D018749:RNA, Plant