Abstract
BackgroundBrucellosis 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.FindingsHere, 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.ConclusionThe 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.
Highlights
Brucellosis is a worldwide anthropozoonotic disease caused by an in vivo intracellular pathogen belonging to genus Brucella
In a technical experiment designed to determine the usefulness of the Genome-directed primers (GDPs) for generation of cDNA from B. melitensis mRNA and subsequent hybridization to microarrays, a primer set (BmGDPs) generated from GDP-Finder software was compared with a commercially available set of random hexamer primers (RHPs)
Each method was used on two identical B. melitensis RNA samples isolated from log phase cultures, and the resulting cDNA was labeled with Cy3 and co-hybridized with Cy5-labeled genomic DNA on two arrays for each primer type
Summary
Brucellosis is a worldwide anthropozoonotic disease caused by an in vivo intracellular pathogen belonging to genus Brucella. The characterization of the transcriptome of intracellular pathogens 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. Gene expression profiles of intramacrophage Salmonella typhimurium, Listeria monocytogenes and Shigella flexneri were studied using this methodology [6,7,8] Another successful approach developed entails lysing host cells with cold water, followed by enzymatic digestion of host genetic material with RNase and DNase while bacteria remain protected by their cell envelope [9]. The method described by Motley et al (2004) produced antisense RNA which could not be used to hybridize on the oligo-arrays [14], and like the Linear Amplification of Prokaryotic Transcripts (LAPT) [15], does not discriminate RNA populations during amplification
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