BioProject

Mycobacteria infect macrophages that aggregate with additional macrophages and lymphocytes to form granulomas. More...

Mycobacteria infect macrophages that aggregate with additional macrophages and lymphocytes to form granulomas. We have used a functional genomics approach to identify immune response genes expressed during granuloma formation in Mycobacterium marinum-infected transparent zebrafish larvae where individual infection steps can be viewed in real time. We assessed RNA expression profiles from zebrafish larvae that were either infected with Mycobacterium marinum, mock-infected, or uninfected. Zebrafish infections were performed at 1 day post-fertilization (dpf), and samples were derived from pools of 6dpf zebrafish larvae. Keywords: host response to infection Overall design: Total RNA was purified from pooled intact zebrafish larvae (31-82 larvae/pool, 3 biological replicate pools/condition) using Trizol reagent (Invitrogen) followed by DNase I digestion (DNA-Free, Ambion) according to manufacturers' protocols. Total RNA from each replicate pool (12ug RNA/replicate) was used as template for independent cDNA synthesis and in vitro transcription reactions (BioArray HighYield RNA Transcript Labeling Kit; Enzo Life Sciences) to generate biotinylated cRNA targets. cRNA targets (30ug/replicate) were fragmented using standard methods. Hybridization and scanning were performed using standard Affymetrix protocol. Raw expression values were normalized (Invariant set method) and modeled (PM-MM model), and present/absent calls were generated using dChip software (build date Dec.11, 2005). Less...