Isolation of high quality RNA and molecular manipulations with various tissues of Populus

Abstract

Populus possesses many characteristics as the model forest species for functional genomic studies, including modest genome size, rapid growth, relative ease of experimental manipulation, and the range of available genetic tools. Isolation of high quality RNA from Populus species and tissues rich in polyphenols and polysaccharides is often difficult. These secondary metabolites not only decrease the yield, but also the quality of RNA is almost unusable. Here we describe a modified procedure to isolate high quality RNA from Populus various tissues, including roots, green stems, leaves, and buds. Major changes are the additional treatments: SDS extraction and ethanol precipitation after CTAB-LiCl purification. Following this procedure, we routinely obtained the high quality RNA, which was suitable for downstream molecular manipulations. Integrity and purity were evaluated by using gel electrophoresis and spectrophotometry (A260/A230 and A260/A280). A260/A230 ratios were greater than 2.0, and the A260/A280 ratios were always between 1.9 and 2.0. The RNA isolated was successfully used for reverse transcription-polymerase chain reaction, rapid amplification of cDNA ends, and suppression subtraction hybridization library construction.