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[…] d using horseradish peroxidase-conjugated secondary antibodies and membranes were revealed with ECL (GE Healthcare Biosciences). Loading controls were checked using an antibody to HSP27 (sc-1048, Santa Cruz; dilution 1∶5,000). The first lane of the blot contained 10 µg of Leishmania lysate, to ensure that the antibodies used in the experiment did not cross-react with Leishmania proteins. Data shown are representative of three independent experiments conducted on MDM derived from three different healthy donors., Analysis of miRNA differentially regulated after L. major infection was carried out for each experimental time point separately using the MultiExperiment Viewer (MeV) v4.7.1 from the TM4 software package available as open-source software at http://www.tm4.org/mev.html., Hierarchical Clustering was performed using the Euclidean distance metric with complete linkage option., For miRNA target identification, we used the miRWalk comprehensive database that provides information on human miRNAs experimentally validated binding sites target genes updated on 15th March 2011., InnateDB database was used to classify all miRNA targets according to their associated GO terms using the hyper-geometric test and the Benjamini Hochberg correction method (default parameters). An enrichment analysis was performed using the BINGO plugIn of Cytoscape v2.8.3 , based on the GO terms revealed by the up- or down-regulated miRNA targets at each time point. We used the hyper-geometric test and the Benjamini Hochberg FDR correction method, and a 0.001 significance level due to the high proportion of associated GO terms. We finally used TransmiR database (updated on 19th March 2012) for transcription factors (TFs) regulating miRNA transcription to identify experimentally validated TFs that are upstream of deregulated miRNAs., The statistical significance of the quantitative differences between the different sample groups was determined by application of Student's two-tailed t test. P values of <0.05 were considered statistically s […]

Pipeline specifications

Software tools TM4, BiNGO, Cytoscape
Databases miRWalk InnateDB