Maurer Michelle, Klassert Tilman E, Löffler Bettina, Slevogt Hortense, Tuchscherr Lorena
Institute for Medical Microbiology, Jena University Hospital, 07747 Jena, Germany.
Department of Respiratory Medicine, Medizinische Hochschule Hannover, 30625 Hannover, Germany.
Microorganisms. 2023 Apr 13;11(4):1020. doi: 10.3390/microorganisms11041020.
evades antibiotic therapy and antimicrobial defenses by entering human host cells. Bacterial transcriptomic analysis represents an invaluable tool to unravel the complex interplay between host and pathogen. Therefore, the extraction of high-quality RNA from intracellular lays the foundation to acquire meaningful gene expression data. In this study, we present a novel and straightforward strategy to isolate RNA from internalized after 90 min, 24 h, and 48 h postinfection. Real-time PCR data were obtained for the target genes and , which play major roles during infection. The commonly used reference genes , , , , and were analyzed under different conditions: bacteria from culture (condition I), intracellular bacteria (condition II), and across both conditions I and II. The most stable reference genes were used for the normalization of and . Delta C (quantification cycle) values had a relatively low variability and thus demonstrated the high quality of the extracted RNA from intracellular during the early phase of infection. The established protocol allows the extraction and purification of intracellular staphylococcal RNA while minimizing the amount of host RNA in the sample. This approach can leverage reproducible gene expression data to study host-pathogen interactions.
通过进入人类宿主细胞来逃避抗生素治疗和抗菌防御。细菌转录组分析是揭示宿主与病原体之间复杂相互作用的宝贵工具。因此，从细胞内提取高质量的RNA为获取有意义的基因表达数据奠定了基础。在本研究中，我们提出了一种新颖且直接的策略，用于在感染后90分钟、24小时和48小时从内化的[细菌名称未给出]中分离RNA。获得了在感染过程中起主要作用的靶基因[基因名称未给出]和[基因名称未给出]的实时PCR数据。在不同条件下分析了常用的参考基因[基因名称未给出]、[基因名称未给出]、[基因名称未给出]、[基因名称未给出]和[基因名称未给出]：来自培养物的细菌（条件I）、细胞内细菌（条件II）以及条件I和条件II两者。使用最稳定的参考基因对[靶基因名称未给出]和[靶基因名称未给出]进行标准化。ΔC（定量循环）值具有相对较低的变异性，因此证明了在感染早期从细胞内[细菌名称未给出]中提取的RNA质量很高。所建立的方案允许提取和纯化细胞内葡萄球菌RNA，同时使样品中宿主RNA的量最小化。这种方法可以利用可重复的基因表达数据来研究宿主 - 病原体相互作用。
