Lewinsohn, E., Institute of Biological Chemistry, Washington State University, Pullman, 99164-6340, WA, United States
Steele, C.L., Institute of Biological Chemistry, Washington State University, Pullman, 99164-6340, WA, United States
Croteau, R., Institute of Biological Chemistry, Washington State University, Pullman, 99164-6340, WA, United States

Stems of woody conifers contain high levels of polysaccharides and phenolic compounds that complicate the isolation of functional RNA from this highly lignified tissue. These difficulties were overcome by pulverizing the frozen tissue with a stainless-steel mortar and by effectively sequestering interfering phenolic compounds with vinylpyrrolidone polymers, thereby minimizing damage to nucleic acids during extraction. RNases were inhibited by aurintricarboxylic acid, and gelatinous polysaccharides were removed by inclusion of a 10% ethanol precipitation step. RNA was then isolated by precipitation with 33% isopropanol and ultracentrifugation onto a cushion of 5.7 M CsCl. Yields of 10 to 20 μg RNA/g FW were obtained from woody stems of several different gymnosperm species, including grand fir (Abies grandis), lodgepole pine (Pinus contorta), loblolly pine (Pinus taeda), Douglas fir (Pseudotsuga menziesii) and Pacific yew (Taxus brevifolia). The high quality of the RNA obtained was determined by UV-spectrophotometry, denaturing agarose gel electrophoresis, and in-vitro translation, and this material was used to construct cDNA libraries. © 1994 Kluwer Academic Publishers.