Selective amplification of Brucella melitensis mRNA from a mixed host-pathogen total RNA View Full Text


Ontology type: schema:ScholarlyArticle      Open Access: True


Article Info

DATE

2010-09-28

AUTHORS

Carlos A Rossetti, Cristi L Galindo, Harold R Garner, L Garry Adams

ABSTRACT

BACKGROUND: Brucellosis is a worldwide anthropozoonotic disease caused by an in vivo intracellular pathogen belonging to genus Brucella. The characterization of brucelae transcriptome's during host-pathogen interaction has been limited due to the difficulty of obtaining an adequate quantity of good quality eukaryotic RNA-free pathogen RNA for downstream applications. FINDINGS: Here, we describe a combined protocol to prepare RNA from intracellular B. melitensis in a quantity and quality suitable for pathogen gene expression analysis. Initially, B. melitensis total RNA was enriched from a host:pathogen mixed RNA sample by reducing the eukaryotic RNA..Then, to increase the Brucella RNA concentration and simultaneously minimize the contaminated host RNA in the mixed sample, a specific primer set designed to anneal to all B. melitensis ORF allows the selective linear amplification of sense-strand prokaryotic transcripts in a previously enriched RNA sample. CONCLUSION: The novelty of the method we present here allows analysis of the gene expression profile of B. melitensis when limited amounts of pathogen RNA are present, and is potentially applicable to both in vivo and in vitro models of infection, even at early infection time points. More... »

PAGES

244-244

Identifiers

URI

http://scigraph.springernature.com/pub.10.1186/1756-0500-3-244

DOI

http://dx.doi.org/10.1186/1756-0500-3-244

DIMENSIONS

https://app.dimensions.ai/details/publication/pub.1030025757

PUBMED

https://www.ncbi.nlm.nih.gov/pubmed/20920187


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