Extraction and analysis of RNA isolated from pure bacteria-derived outer membrane vesicles

Janine Habier, Patrick May, Anna Heintz-Buschart, Anubrata Ghosal, Anke K. Wienecke-Baldacchino, Esther N.M. Nolte-‘t Hoen, Paul Wilmes, Joëlle V. Fritz*

*Corresponding author for this work

Research output: Chapter in Book/Report/Conference proceedingChapterpeer-review

11 Citations (Scopus)

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.

Original languageEnglish
Title of host publicationMethods in Molecular Biology
PublisherHumana Press Inc.
Pages213-230
Number of pages18
DOIs
Publication statusPublished - 2018
Externally publishedYes

Publication series

NameMethods in Molecular Biology
Volume1737
ISSN (Print)1064-3745

Keywords

  • Analysis
  • Bacteria
  • Density gradient
  • Extraction
  • Gram-negative
  • Outer membrane vesicle (OMV)
  • RNA
  • Sequencing
  • Ultracentrifugation
  • Ultrafiltration

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