Rossetti Carlos A, Galindo Cristi L, Garner Harold R, Adams L Garry
Department of Veterinary Pathobiology, College of Veterinary Medicine, Texas A&M University, College Station, TX, USA.
BMC Res Notes. 2010 Sep 28;3:244. doi: 10.1186/1756-0500-3-244.
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.
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.
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.
布鲁氏菌病是一种由布鲁氏菌属的体内细胞内病原体引起的全球性人畜共患病。由于难以获得足够数量的高质量无真核RNA的病原体RNA用于下游应用，布鲁氏菌在宿主-病原体相互作用过程中转录组的特征研究受到限制。
在此，我们描述了一种组合方案，用于从胞内马尔他布鲁氏菌中制备数量和质量适合病原体基因表达分析的RNA。首先，通过减少真核RNA，从宿主：病原体混合RNA样本中富集马尔他布鲁氏菌总RNA。然后，为了提高布鲁氏菌RNA浓度并同时最大限度地减少混合样本中受污染的宿主RNA，设计了一组与所有马尔他布鲁氏菌开放阅读框退火的特异性引物，用于在先前富集的RNA样本中对有义链原核转录本进行选择性线性扩增。
我们在此介绍的方法的新颖之处在于，当病原体RNA数量有限时，能够分析马尔他布鲁氏菌的基因表达谱，并且甚至在感染早期时间点，也可能适用于体内和体外感染模型。
