) was applied to bring the histograms of all microarrays into the same scale. Technical replicates were averaged. Differentially expressed genes between the strains were detected by applying t-tests with a Benjamini and Hochberg adjusted p-value correction. RT-qPCR RT-qPCR reactions were
performed as described by Santangelo et al. [13, click here 20] using DNA-free RNA (1 μg) extracted from mid-exponential growth-phase cultures and specific primers. Relative quantification was performed by using sigA as a reference gene and a subsequent analysis for statistical significance of the derived results was performed by using the Pair Wise Fixed Reallocation Randomization test [21]. The mean value of PCR efficiency for the primers (Additional file 2: Table S2) was 92% to 100%. These values were calculated using both the classical dilution curve and slope calculation (E = 10 [−1/slope] − 1) [21] and an estimation by absolute fluorescence increase [22]. Acknowledgements We acknowledge The Wellcome Trust for funding BuG@S (Bacterial Microarray Group at St George’s, University of London) for supply of the microarray and associated support. We are grateful to Julia Sabio y García for her technical assistance in the confocal experiments. We
also thank the group of Dr. Jacobs Jr WR for the specialized transduction system provided. The present study was supported by NIH/NIAID 1R01AI083084. Experiments with animals were funded by INTA grant PE PNBIO 1131034 and ANCyPT grant PICT 1103. MP Santangelo and F. Bigi are CONICET fellows. FB and MGG are supported by a cooperation grant from Ministry of GSK1838705A purchase Science MI-503 and Technology (MinCyT-Argentina) and International Buro of the Federal Ministry of Education and Research (Germany). Electronic supplementary material Additional file 1: Table S1: Differential expressed genes between MtΔmce2R/M. tuberculosis H37Rv. (DOCX 57 KB) Additional file 2: Table S2: Primers used in RT-qPCR. (DOCX 41 KB) References 1. Glickman MS, Jacobs WR Jr: Microbial pathogenesis of Mycobacterium tuberculosis: dawn of a discipline.
Cell 2001, 104:477–485.PubMedCrossRef 2. Hingley-Wilson SM, Sambandamurthy VK, Jacobs WR Jr: Survival perspectives from the world’s most successful pathogen, Mycobacterium tuberculosis. Nat Immunol 2003, 4:949–955.PubMedCrossRef 3. Arruda S, Bomfim G, Knights R, Huima-Byron T, Riley LW: Cloning G protein-coupled receptor kinase of an M. tuberculosis DNA fragment associated with entry and survival inside cells. Science 1993, 261:1454–1457.PubMedCrossRef 4. Casali N, Riley LW: A phylogenomic analysis of the Actinomycetales mce operons. BMC Genomics 2007, 8:60.PubMedCrossRef 5. Flesselles B, Anand NN, Remani J, Loosmore SM, Klein MH: Disruption of the mycobacterial cell entry gene of Mycobacterium bovis BCG results in a mutant that exhibits a reduced invasiveness for epithelial cells. FEMS Microbiol Lett 1999, 177:237–242.PubMedCrossRef 6. Sassetti CM, Rubin EJ: Genetic requirements for mycobacterial survival during infection.