Waddell Simon J, Butcher Philip D
Medical Microbiology, Division of Cellular & Molecular Medicine, Centre for Infection, St George's University of London, London, UK.
Methods Mol Biol. 2010;642:75-91. doi: 10.1007/978-1-60327-279-7_6.
Analysis of the transcriptional profiles of Mycobacterium tuberculosis after treatment with antimycobacterial compounds has improved our understanding of the ways mycobacteria respond to antibiotic stress, and revealed new insights into the mode of action of different antimycobacterial compound classes. RNA profiling of drug-induced changes has become an important tool in multiple stages of the antibacterial drug development process from target elucidation, to identifying target drift, and ultimately to revealing drug resistance mechanisms. The transcriptional response of M. tuberculosis to antimycobacterial compounds may be determined in isolation, in comparison with other compound classes, or between drug-sensitive and drug-resistant mycobacterial isolates. Additional information confirming the growth state of mycobacteria on addition of the antibacterial compound, and the effect that this compound has on mycobacterial growth, is essential for interpreting the transcriptional signatures acquired. This chapter describes the methods required for the extraction of representative total mycobacterial RNA, the subsequent hybridisation of this RNA to an M. tuberculosis complex microarray, and the analysis strategies employed to interpret the transcriptional data generated.
对抗分枝杆菌化合物处理后的结核分枝杆菌转录谱进行分析，增进了我们对分枝杆菌应对抗生素应激方式的理解，并揭示了不同类抗分枝杆菌化合物作用模式的新见解。药物诱导变化的RNA谱分析已成为抗菌药物开发过程多个阶段的重要工具，从靶点阐明到识别靶点漂移，最终揭示耐药机制。结核分枝杆菌对抗分枝杆菌化合物的转录反应可以单独确定，与其他化合物类别进行比较，或者在药物敏感和耐药分枝杆菌分离株之间进行确定。确认添加抗菌化合物时分枝杆菌生长状态以及该化合物对分枝杆菌生长的影响的额外信息，对于解释获得的转录特征至关重要。本章描述了提取代表性分枝杆菌总RNA所需的方法、该RNA随后与结核分枝杆菌复合微阵列的杂交以及用于解释所产生转录数据的分析策略。
