A Method for Enzymic Extraction and the Measurement of Chloroplast RNA

Abstract

A method is described for measuring RNA associated with chloroplast thylakoid membranes. Washed thylakoids are incubated with ribonucleases A and T(1), under low Mg(2+) conditions, to release hydrolyzed RNA into solution. After removing the membranes by centrifugation, the mono- and oligonucleotides are adsorbed by Dowex 1-X2 in miniature columns made from Pasteur pipettes, and then eluted with 2 n HCl. RNA is estimated from the absorbance of the eluate at 260 nanometers, with corresponding values obtained by the orcinol reaction for pentose. Polyacrylamide gel electrophoresis patterns of extracted RNA indicate that our current procedures for preparation of thylakoids results in material containing variable and often significant levels of RNA from 80S ribosomes. Thus values for total RNA cannot be used as a valid estimate for the level of 70S ribosomes associated with these membranes, unless an additional procedure is used to estimate the per cent contamination by 80S ribosomes.Recoveries of digested RNA from the Dowex resin of 94 to 98% were obtained with 2 milliliters of HCl eluant, making possible the analysis of thylakoid samples with as little as 4 micrograms of RNA. The procedure involves small columns and only one centrifugation, so that it is useful for obtaining reliable measurements from multiple samples.