Extraction and Purification of Outer Membrane Vesicles and Their Associated RNAs

Outer membrane vesicles (OMVs), produced by Gram negative-bacteria and sRNAs, are key players in cell-to-cell communication and interactions of bacteria with the environment. OMVs act as information carriers and encapsulate various molecules such as proteins, lipids, metabolites, and RNAs. OMVs and sRNAs play a broad range of functions from pathogenesis to stress resistance, to biofilm formation and both mediate interkingdom signaling. Various studies indicate that there is a mechanism of intercellular communication mediated by OMV-derived bacterial RNAs that is conserved among certain bacterial species. Here we describe methods for the extraction and purification of vesicles produced by Gram-negative bacteria, such as Pseudomonas brassicacearum and Escherichia coli, and address methods for the extraction of OMVs-derived sRNA and techniques for the analysis of sRNAs.