Transcriptomics of mycobacterial microaggregates

During initial colonization of the airways, MAH form microaggregates composed of 3-20 bacteria on human respiratory epithelial cells, which provides an environment for phenotypic changes leading to efficient mucosal invasion. DNA microarray was employed to identify genes associated with the microaggregate phenotype. Bacteria were incubated with Hep-2 epithelial cells for 24 hrs to form microaggregates or incubated in tissue culture media alone as the control (planktonic bacteria). Bacterial RNA was isolated, purified using MicrobeEnrich, and amplified using Abmbion's Bacterial MessageAMP kit. RNA was hybridized to Affymetrix custom made mycobacterium avium 104 microarrays在气道初始定植阶段，鸟分枝杆菌（Mycobacterium avium, MAH）会在人类呼吸道上皮细胞表面形成由3至20个细菌构成的微聚集体，该结构可为引发高效黏膜侵袭的表型改变提供适宜环境。本研究采用DNA微阵列（DNA microarray）技术，筛选与该微聚体表型相关的基因。实验中将细菌与Hep-2上皮细胞共孵育24小时以诱导微聚集体形成，同时设置仅在组织细胞培养基中孵育的浮游细菌对照组。随后分离细菌RNA，使用MicrobeEnrich试剂盒完成纯化，并通过Abmbion公司的Bacterial MessageAMP试剂盒进行扩增。最终将扩增后的RNA与Affymetrix定制的鸟分枝杆菌104微阵列进行杂交实验。