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

doi:10.1007/978-1-4939-7634-8_13

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

Luxembourg Centre for Systems Biomedicine, University of Luxembourg, Belvaux, Luxembourg.
Department of Soil Ecology, Helmholtz-Centre for Environmental Research GmbH (UFZ), Halle (Saale), Germany.
Department of Biology, Massachusetts Institute of Technology , Cambridge , MA , USA.
Life Sciences Research Unit, University of Luxembourg, Belvaux, Luxembourg.
Centre Hospitalier Luxembourg, Luxembourg, Luxembourg. [email protected].

Research output: Chapter in Book/Report/Conference proceeding › Chapter › Academic › peer-review

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 language	English
Title of host publication	Bacterial Regulatory RNA
Subtitle of host publication	Methods and Protocols
Editors	Véronique Arluison, Claudio Valverde
Publisher	Springer
Chapter	13
Pages	213-230
Number of pages	18
ISBN (Electronic)	978-1-4939-7634-8
ISBN (Print)	978-1-4939-7633-1
DOIs	https://doi.org/10.1007/978-1-4939-7634-8_13
Publication status	Published - 2018

Publication series

Name	Methods in Molecular Biology
Volume	1737
ISSN (Print)	1064-3745

Keywords

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

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10.1007/978-1-4939-7634-8_13

ExtractionFinal published version, 589 KBLicence: Taverne

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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 (pp. 213-230). (Methods in Molecular Biology; Vol. 1737). Springer. https://doi.org/10.1007/978-1-4939-7634-8_13

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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",

author = "Janine Habier and Patrick May and Anna Heintz-Buschart and Anubrata Ghosal and Wienecke-Baldacchino, {Anke K} and {Nolte-'t Hoen}, {Esther N M} and Paul Wilmes and Fritz, {Jo{\"e}lle V}",

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doi = "10.1007/978-1-4939-7634-8_13",

language = "English",

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series = "Methods in Molecular Biology",

publisher = "Springer",

pages = "213--230",

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Habier, J, May, P, Heintz-Buschart, A, Ghosal, A, Wienecke-Baldacchino, AK, Nolte-'t Hoen, ENM, Wilmes, P & Fritz, JV 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

Extraction and Analysis of RNA Isolated from Pure Bacteria-Derived Outer Membrane Vesicles. / Habier, Janine; May, Patrick; Heintz-Buschart, Anna et al.
Bacterial Regulatory RNA: Methods and Protocols. ed. / Véronique Arluison; Claudio Valverde. Springer, 2018. p. 213-230 (Methods in Molecular Biology; Vol. 1737).

Research output: Chapter in Book/Report/Conference proceeding › Chapter › Academic › peer-review

TY - CHAP

T1 - Extraction and Analysis of RNA Isolated from Pure Bacteria-Derived Outer Membrane Vesicles

AU - Habier, Janine

AU - May, Patrick

AU - Heintz-Buschart, Anna

AU - Ghosal, Anubrata

AU - Wienecke-Baldacchino, Anke K

AU - Nolte-'t Hoen, Esther N M

AU - Wilmes, Paul

AU - Fritz, Joëlle V

PY - 2018

Y1 - 2018

N2 - 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.

AB - 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.

KW - Bacteria

KW - RNA

KW - Outer membrane vesicle (OMV)

KW - Gram-negative

KW - Sequencing

KW - Analysis

KW - Extraction

KW - Ultracentrifugation

KW - Ultrafiltration

KW - Density gradient

U2 - 10.1007/978-1-4939-7634-8_13

DO - 10.1007/978-1-4939-7634-8_13

M3 - Chapter

C2 - 29484596

SN - 978-1-4939-7633-1

T3 - Methods in Molecular Biology

SP - 213

EP - 230

BT - Bacterial Regulatory RNA

A2 - Arluison, Véronique

A2 - Valverde, Claudio

PB - Springer

ER -

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

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