Extraction and Analysis of RNA Isolated from Pure Bacteria-Derived Outer Membrane Vesicles
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.
KeywordsBacteria RNA Outer membrane vesicle (OMV) Gram-negative Sequencing Analysis Extraction Ultracentrifugation Ultrafiltration Density gradient
This work was supported by a CORE programme grant (CORE/14/BM/8066232) to J.V.F and by a Proof-of-Concept grant (PoC/13/02) to P.W, all funded by the Luxembourg National Research Fund (FNR). We are grateful to Dr. Jean-François Ménétret from the Department of Structural Biology and Genomics Institute of Genetics and of Molecular and Cellular Biology (IGBMC; France) for the acquisition of the electron microscopy images presented in this protocol. We also thank Alton Etheridge and David Galas (Pacific Northwest Diabetes Research Institute, Seattle, Washington 98122) for RNA-Seq. Bioinformatics analyses presented in this book chapter were carried out in part using the HPC facilities of the University of Luxembourg (http://hpc.uni.lu).
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