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Extraction and Analysis of RNA Isolated from Pure Bacteria-Derived Outer Membrane Vesicles
Habier, Janine; May, Patrick; Heintz-Buschart, Anna et al.
2018In Arluison, Véronique; Valverde, Claudio Valverde (Eds.) Bacterial Regulatory RNA
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Keywords :
Bacteria; RNA; Outer membrane vesicle (OMV); Gram-negative; Sequencing; Extraction; Ultracentrifugation; Ultrafiltration; Density gradient
Abstract :
[en] 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.
Research center :
- Luxembourg Centre for Systems Biomedicine (LCSB): Eco-Systems Biology (Wilmes Group)
- Luxembourg Centre for Systems Biomedicine (LCSB): Bioinformatics Core (R. Schneider Group)
ULHPC - University of Luxembourg: High Performance Computing
Disciplines :
Microbiology
Author, co-author :
Habier, Janine ;  University of Luxembourg > Luxembourg Centre for Systems Biomedicine (LCSB)
May, Patrick  ;  University of Luxembourg > Luxembourg Centre for Systems Biomedicine (LCSB)
Heintz-Buschart, Anna
Ghosal, Anubrata
Wienecke-Baldacchino, Anke ;  University of Luxembourg > Faculty of Science, Technology and Communication (FSTC) > Life Science Research Unit
Nolte-'t Hoen, Esther N.M
Wilmes, Paul ;  University of Luxembourg > Luxembourg Centre for Systems Biomedicine (LCSB)
Fritz, Joëlle ;  University of Luxembourg > Luxembourg Centre for Systems Biomedicine (LCSB)
External co-authors :
yes
Language :
English
Title :
Extraction and Analysis of RNA Isolated from Pure Bacteria-Derived Outer Membrane Vesicles
Publication date :
27 February 2018
Main work title :
Bacterial Regulatory RNA
Author, co-author :
Arluison, Véronique
Valverde, Claudio Valverde
Publisher :
Humana Press, New York, United States
Edition :
Methods in Molecular Biology
ISBN/EAN :
978-1-4939-7634-8
Collection name :
1737
Pages :
213-230
Peer reviewed :
Peer reviewed
Focus Area :
Systems Biomedicine
FnR Project :
FNR8066232 - Host-microbes Interactions Via Bacterial Extracellular Small Rnas, 2014 (01/04/2015-31/08/2017) - Joelle Fritz
Available on ORBilu :
since 28 February 2018

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