Extraction and Analysis of RNA Isolated from Pure Bacteria-Derived Outer Membrane Vesicles
Publication date
2018
Authors
Habier, Janine
May, Patrick
Heintz-Buschart, Anna
Ghosal, Anubrata
Wienecke-Baldacchino, Anke K
Nolte - t Hoen, Esther
Wilmes, Paul
Fritz, Joëlle V
Editors
Arluison, Véronique
Valverde, Claudio
Advisors
Supervisors
Document Type
Part of book
Metadata
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License
taverne
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.
Keywords
Bacteria, RNA, Outer membrane vesicle (OMV), Gram-negative, Sequencing, Analysis, Extraction, Ultracentrifugation, Ultrafiltration, Density gradient, Taverne
Citation
Habier, J, May, P, Heintz-Buschart, A, Ghosal, A, Wienecke-Baldacchino, A K, Nolte-'t Hoen, E N M, Wilmes, P & Fritz, J V 2018, Extraction and Analysis of RNA Isolated from Pure Bacteria-Derived Outer Membrane Vesicles. in V Arluison & C Valverde (eds), Bacterial Regulatory RNA : Methods and Protocols. Methods in Molecular Biology, vol. 1737, Springer, pp. 213-230. https://doi.org/10.1007/978-1-4939-7634-8_13