A rapid method to quantitate non-labeled RNA species in bacterial cells

Abstract We have developed a rapid method to quantitate specific bacterial RNA species. The method measures the steady-state level of RNA, produces a linear response over more than a 16-fold range of RNA concentration, and can be used for Staphylococcus aureus , Escherichia coli and Bacillus subtilis . In this method, a sheared whole-cell lysate of approx. 7 × 10 8 organisms, prepared as for plasmid screening, is separated on agarose, blotted to a nitrocellulose filter, hybridized with a radiolabeled DNA probe, and autoradiographed. The RNA species are quantitated by counting the radioactive bands on the filter. We have applied the method to the measurement of mRNA induction of the genes encoding β-lactamase, ermC rRNA methylase, and the α-complementing fragment of β-galactosidase. Upon induction, a ten-fold increase in the mRNA for each gene was observed. The peak mRNA level occurred after 30 min for β-lactamase, 20 min for β-galactosidase, and 5 min for the ermC rRNA methylase.