Improved Method for Purification of RNA from Stem Tissue of Grapevine and Its Use in mRNA Profiling

Abstract

A procedure that does not involve an ultracentrifugation step for the isolation of functional RNA from recalcitrant grape stem tissue derived from both in vitro and in vivo sources has been optimized. The procedure used an extraction buffer containing a reducing agent, nuclease inhibitor, and mild detergents. RNA was purified from abundant carbohydrates, polyphenols, and other contaminating compounds with the use of insoluble polyvinylpyrrolidone, two cycles of potassium acetate precipitation, and lithium chloride differential dissolution. RNA appeared intact with prominent 28S and 18S rRNA bands, OD_260/280 values ranging from 1.8 to 2.0, and yield ranging from 80 to 120 µg g^-1 tissue. RNA was successfully used in mRNA differential display studies involving reverse transcription, polymerase chain reaction-amplification of cDNA, Northern hybridization, and cDNA probing to elucidate differential gene expression during rooting inductive phase. The procedure worked well for other tissues including leaf, petiole, root, and new shoots.