Zebrafish response to Mycobacterium marinum infection (Compugen series)

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 or mock-infected. 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 Total RNA was purified from pooled intact zebrafish larvae (154-180 larvae/pool, 2 biological replicate pools/condition) and digested with DNase I using an Absolutely RNA Microprep Kit (Stratagene). RNA quality was confirmed using an Agilent 2100 Bioanalyzer. Total RNA from each replicate pool (5 μg/replicate) was used as template for independent reverse transcription reactions utilizing the 3DNA Array 350 Kit (Genisphere) and modified oligo-dT primers containing a 5’ fluorophore/dendrimer-specific sequence. Competing cDNA samples for each hybridization were paired, purified, and hybridized to epoxy-coated glass slides (MWG Biotech) containing the Zebrafish Oligonucleotide Library (Compugen, Sigma-Genosys). Cy3 and Cy5 labeling of respective dendrimer-specific sequences was performed using using the complementary capture reagents provided in the 3DNA Array 350 Kit (Genisphere). Raw Cy3 and Cy5 expression values were normalized (LOWESS) and expression ratios were transformed to log2 values using ScanArray software (Perkin Elmer).