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Author:
Date:
2015-11-20
[Abstract] Studying the transcriptome of bacterial pathogens during infection is a very informative and effective tool for discovering genes that contribute to successful infection. However, isolating bacterial RNA from infected cells or tissues is a challenging process due to the much higher amounts of host RNA in the lysates of infected cells. We have optimized a method for isolating RNA of Listeria monocytogenes (L. monocytogenes) bacteria infecting bone marrow derived macrophage cells (BMDM). After infection, we lyse the cells and filter the lysates through 0.45 µm filters to discard most of the host proteins and RNA. Next, we resuspend the bacteria and extract RNA following DNase treatment. The extracted RNA is suitable for gene expression analysis by real-time PCR or ...
[摘要] 在感染期间研究细菌病原体的转录组是一个非常有益的和有效的工具，用于发现有助于成功感染的基因。然而，从感染的细胞或组织中分离细菌RNA是一个挑战性的过程，因为感染细胞裂解物中宿主RNA的量高得多。我们已经优化了用于分离感染骨髓来源的巨噬细胞（BMDM）的单核细胞增生性李斯特菌（（单核细胞增生李斯特氏菌）细菌）的RNA的方法。感染后，我们裂解细胞并通过0.45μm过滤器过滤裂解物以丢弃大多数宿主蛋白和RNA。接下来，我们重新悬浮细菌，并在DNase处理后提取RNA。提取的RNA适合于通过实时PCR或微阵列的基因表达分析。我们已经在我们对感染期间的单核细胞增生李斯特氏菌基因调节的体外研究中成功地采用了该方案（Lobel等人，2015; Lobel >，2012; Kaplan Zeevi等人，2013; Rabinovich等人，2012）。
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