Cy5-labeled cDNA from the BALF-incubated malT mutant, and (3) Cy3-labeled cDNA from the BHI-incubated wild-type organism vs. Cy5-labeled

cDNA from BHI-incubated malT mutant. Four replications, including dye-swaps, were carried out for each type of hybridization. cDNA was synthesized in the presence of amino-allyl-dUTP (Sigma-Aldrich, St. Louis MO, US), random octamer primers (Biocorps, Montreal, QC, PF-6463922 Canada), SuperScript II transcriptase buy BIBW2992 (Invitrogen, Carlsbad, CA, US), and the RNA (15 μg per reaction) obtained from the BALF- and BHI-incubated organisms, according to the method described by Carrillo et al. [37]. Labeling of the cDNA was carried out indirectly with one of the mono-functional NHS-ester dyes Cy3 or Cy5 (GE Healthcare, CFTRinh-172 order Buckinghamshire, UK), which binds to the amino-allyl-dUTP of the cDNA. The dye labeling efficiency of cDNA was determined spectrophotometrically. The data were submitted

to the Gene Expression Omnibus (GEO: GSE13006). Microarray data analysis Microarray image and data analysis was carried out using the TM4 Suite of software [38] for microarray analysis, (J. Craig Venter Institute, JCVI, USA) as described elsewhere [36]. Briefly, images were analyzed with Spotfinder v3.1.1. The final intensity of each spot was obtained by subtracting the background intensity from the integral spot intensity (the sum of the intensities of all the spot pixels excluding the saturated ones). The spots with intensities less

than one standard deviation above their spot background intensities were eliminated from the downstream analysis, as were the ones with total intensity less than 10000. Replicate spots were analyzed subsequent to the normalization of the data using the LOWESS (locally weighted linear regression) algorithm. The genes that were thus represented by good quality spots (defined by a score assigned by the software based on the number of unsaturated pixels, shape, and signal to noise ratio of the spot) on a minimum of two replicate slides were considered for the downstream analysis using SAM (significance analysis of microarray) to identify the differentially through expressed genes. The differentially expressed genes were classified depending upon their biological roles into various functional categories according to the JCVIs Comprehensive Microbial Resources (CMR) database. Quantitative real-time PCR The parameters of RNA capacity, optimum primer concentration, and the gene dynamic ranges were determined before carrying out the real-time PCR for the relative quantification of the target gene expression. As an endogenous control, the level of prolyl-tRNA-synthetase gene (syp) expression was used to normalize the target gene expression levels, since this gene exhibited the least variation in expression across various conditions in both the microarray and real-time PCR experiments.