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首页> 外文期刊>Parasitology >Gene expression profiling of Leishmania (Leishmania) donovani: overcoming technical variation and exploiting biological variation.
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Gene expression profiling of Leishmania (Leishmania) donovani: overcoming technical variation and exploiting biological variation.

机译：利什曼原虫（Leishmania）donovani的基因表达谱：克服技术差异并利用生物学差异。

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摘要

Gene expression profiling is increasingly used in the field of infectious diseases for characterization of host, pathogen and the nature of their interaction. The purpose of this study was to develop a robust, standardized method for comparative expression profiling and molecular characterization of Leishmania donovani clinical isolates. The limitations and possibilities associated with expression profiling in intracellular amastigotes and promastigotes were assessed through a series of comparative experiments in which technical and biological parameters were scrutinized. On a technical level, our results show that it is essential to use parasite harvesting procedures that involve minimal disturbance of the parasite's environment in order to 'freeze' gene expression levels instantly; this is particularly a delicate task for intracellular amastigotes and for specific 'sensory' genes. On the biological level, we demonstrate that gene expression levels fluctuate during in vitro development of both intracellular amastigotes and promastigotes. We chose to use expression-curves rather than single, specific, time-point measurements to capture this biological variation. Intracellular amastigote protocols need further refinement, but we describe a first generation tool for high-throughput comparative molecular characterization of patients' isolates, based on the changing expression profiles of promastigotes during in vitro differentiation.
机译：基因表达谱在传染病领域中越来越多地用于表征宿主，病原体及其相互作用的性质。这项研究的目的是开发一种强大的，标准化的方法，用于对多形利什曼原虫临床分离株进行比较表达谱分析和分子表征。通过一系列比较实验评估了与胞内变形虫和前鞭毛虫中的表达谱相关的局限性和可能性，在该实验中，对技术和生物学参数进行了仔细研究。从技术上讲，我们的结果表明，必须使用对寄生虫环境造成最小干扰的寄生虫收获程序，才能立即“冻结”基因表达水平。对于细胞内的变形虫和特定的“感觉”基因而言，这尤其是一项微妙的任务。在生物学水平上，我们证明了基因表达水平在细胞内无鞭毛体和前鞭毛体的体外发育过程中会发生波动。我们选择使用表达曲线而不是单个的特定时间点测量来捕获这种生物学差异。细胞内的鞭毛体协议需要进一步完善，但我们描述了基于前体在体外分化过程中表达谱变化的第一代工具，可对患者分离株进行高通量比较分子表征。

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