Reactions were subsequently purified with PCR Purification columns (Qiagen, Valencia, CA) using a modified wash (5 mM KPO4 (pH 8.0) and 80% ethanol) and incremental elution with 4 mM KPO4, pH 8.5. Alexa-Fluor 555 (Invitrogen, Carlsbad, CA) was coupled to the RNA-derived cDNA following the procedure outlined in the BioPrime® Plus Array CGH Indirect Genomic Labeling System (Invitrogen, Carlsbad, CA) and purified using PCR purification columns (Qiagen, Valencia,

CA). Labeled RNA samples were dried completely and re-suspended in ddH2O immediately before hybridization to the microarrays. Microarray construction Unique 70-mer oligonucleotides (Sigma, St. Louis, MO) representing 3,227 ORFs of B. melitensis 16M and unique sequences from B. abortus and B. suis were suspended

in 3× SSC (Ambion, Austin, TX) at 40 μM. The oligonucleotides were spotted in quadruplicate onto ultraGAP glass slides (Corning, Corning, NY) by a custom-built robotic Selleckchem VS-4718 arrayer (Magna Arrayer) assembled at Dr. Stephen A. Johnston’s lab at the University of Texas Southwestern Medical Center (Dallas, TX). The printed slides were steamed, UV cross-linked, and stored in a desiccator until use. Microarray pre-hybridization, hybridization and washing Printed slides were submerged in 0.2% SDS for 2 minutes and washed 3× in ddH2O before incubation in prehybridization solution (5× SSC, 0.1% SDS and 1% BSA) at 45°C for 45 minutes. Next, slides were washed 5× in ddH2O, rinsed with isopropanol, and immediately GDC-0994 dried by centrifugation at 200 × g for 2 minutes at room temperature. The labeled cDNA mix was combined with 1× hybridization buffer (25% formamide, 1× SSC and 0.1%SDS) and applied

to the microarray in conjunction with a 22 × 60 mm LifterSlip (Erie Scientific, 17-DMAG (Alvespimycin) HCl Portsmouth, NH). The microarray slides were hybridized at 42°C for approximately 21 hours in a sealed hybridization chamber with moisture (Corning, Corning, NY), and subsequently washed at room CDK inhibitor temperature with agitation in 2× SSC and 0.2% SDS (pre-heated to 42°C) for 10 minutes, 5 minutes in 2× SSC, followed by 0.2× SSC for 5 minutes, and dried by centrifugation for 2 minutes (200 × g) at room temperature. Microarray data acquisition and analysis Array slides were scanned using GenePix 4100A (Molecular Devices, Sunnyvale, CA) and GenePix 6.1 Pro software. Seralogix, Inc. (Austin, TX) performed microarray analysis, normalizing the data and identifying differentially expressed genes by a two-tail z-score level greater than ± 1.96, equating to a confidence level of 95%. Additionally, the NIH/NIAID WRCE bioinformatics core performed microarray analysis as follows: GeneSifter (VizX Labs, Seattle, WA) was used to perform normalization based on the global mean and genes with alterations of least a 1.5-fold, with a p value of 0.05 or less based on Student’s t-test were deemed as statistically significant.