Isolation of RNA from mycobacteria grown under in vitro and in vivo conditions

Isolation of RNA from mycobacteria is very difficult to perform, and the yi elds are generally very lon. We describe an approach to isolate RNA from my cobacterial species which combines the disruption of mycobacterial cells by a silica/ceramic matrix in a reciprocal shaker with the ease and efficienc y of subsequent RNA purification on spin columns with silica gel-based memb ranes. This method is rapid, easy to perform and yields high amounts of pur e, intact total RNA. Due to its safety, this method is applicable even to g roup 3 biological hazard organisms like Mycobacterium tuberculosis. By comb ining a method for the isolation of phagosomal bacteria from infected prima ry macrophages with the novel RNA isolation technique, we are able to monit or gene expression during infection even in bacteria which are rather resis tant to generic manipulation, like Mycobacterium bovis. (C) 2000 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.