Total Bacterial RNA Isolation and Northern Blotting Analysis.

Abstract

The study of bacterial gene expression during infection provides vital information for researchers to understand bacterial pathogenesis and infection. The ability to obtain clean and undegraded RNA could be challenging and daunting and remains the most crucial experimental step prior to downstream analyses, such as Northern blotting, quantitative PCR (qPCR), and RNA-seq.This chapter describe two methods (acid guanidinium thiocyanate (TRIzol) phenol-chloroform and hot phenol) commonly used to isolate total bacterial RNA and are suitable for both Gram-positive and Gram-negative bacteria. Procedures such as RNA quantification and DNase treatment are also included to ensure amount and quality of the RNA samples. The second part of the chapter includes a method used to analyze bacterial gene expression (Northern blotting), two methods to generate radioactive probes, as well as target detection using a phosphorimager.