Extraction and Analysis of RNA Isolated from Pure Bacteria-Derived Outer Membrane Vesicles

Methods in Molecular Biology
Janine HabierJoëlle V Fritz

Abstract

Outer membrane vesicles (OMVs) are released by commensal as well as pathogenic Gram-negative bacteria. These vesicles contain numerous bacterial components, such as proteins, peptidoglycans, lipopolysaccharides, DNA, and RNA. To examine if OMV-associated RNA molecules are bacterial degradation products and/or are functionally active, it is necessary to extract RNA from pure OMVs for subsequent analysis. Therefore, we describe here an isolation method of ultrapure OMVs and the subsequent extraction of RNA and basic steps of RNA-Seq analysis. Bacterial culture, extracellular supernatant concentration, OMV purification, and the subsequent RNA extraction out of OMVs are described. Specific pitfalls within the protocol and RNA contamination sources are highlighted.

Citations

Oct 5, 2018·Annual Review of Animal Biosciences·Janos ZempleniEzra Mutai
May 7, 2020·Experimental Biology and Medicine·Heon-Jin Lee
Jan 8, 2021·Frontiers in Immunology·Camille Martin-GallausiauxPaul Wilmes

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