Preparation of RNA

Abstract

The preparation of high quality (i.e., intact) total RNA from biological samples 1s the primary step in the study of gene expression. It is the procedure that bridges the interface between the experimental manipulation of the hvmg system, and the subsequent analysis of effects through molecular techniques. Arabidopsis is an ideal system for such analyses. Large quantities of plant material are easily obtained, from which mrlligram quantities of total RNA may be isolated. Conversely, small scale preparations from one or a few plants can be used to rapidly obtain multiple experimental samples. The procedure, a variation of a method origmally proposed by Palmiter (I), uses detergent sodium dodecyl sulfate (SDS) and phenol for lysrs and denaturation of cellular constituents. The predommant alternative method of Chirgwm et al (2) employs guamdmium tsothiocyanate as a denaturant. This latter method has also spawned variations, most notably a single‐step method that ehmmates an ultracentrifugation step (3). Although this method may compare favorably with the procedures presented herein with regard to technical simplicity, our protocol consistently results m high yields of intact RNA of a quality suitable for such manipulations as RNA blot hybridizations, RACE polymerase chain reaction (PCR), in vrtro translation, RNA sequencing and cDNA cloning,