melitensis 16 M grown in tryptic soy broth (TSB) (BD) was washed with 25 ml of J-buffer [0.1 M Tris pH 8.0; 0.1 M EDTA; 0.15 M NaCl] and then lysed in 1 ml of J-buffer containing 10% lysozyme solution

(10 mg/ml in 0.25 M Tris, pH 8.0). After 10 min of incubation, DNA was released from the cells by sodium N-lauroyl sarcosine (Sigma) treatment followed by degradation of RNA by DNase-free RNase (Roche Applied Science, Indianapolis, IN) treatment and digestion of proteins with proteinase K (Roche Applied Science). The resulting solution was transferred to a dialysis bag and dialyzed against TE [10 mM Tris, pH 8.0 and 1 mM EDTA] overnight at 37°C. DNA was subsequently extracted twice using neutral water-saturated phenol (Ambion) first and then MK 8931 concentration ether (Sigma) before dialyzing overnight against TE. DNA concentration was quantified by NanoDrop® ND-1000 (NanoDrop) and stored at 4°C until used. B. melitensis genomic DNA was labeled overnight by directed incorporation of Cy5-dCTP (Amersham

Pharmacia Biosciences, Piscataway, NJ) using random primers solution and Klenow fragment from the BioPrime DNA labeling system kit (Invitrogen, Carlsbad, CA) and 50× dNTPs (1:2 dCTP) (Invitrogen). The reaction was stopped by adding 5 μl of stop buffer from the BioPrime kit, and unincorporated Cy5 dye was removed using a PCR purification kit (Qiagen, Valencia, CA). The labeled DNA was eluted in 1 mM Tris pH 8.0 and kept in the dark at 4°C until used. Construction of cDNA microarrays A set of MEK inhibitor clinical trial unique 70-base Low-density-lipoprotein receptor kinase oligonucleotides www.selleckchem.com/products/rg-7112.html representing 3,227 ORFs of B. melitensis strain 16 M plus unique/divergent genes from B. abortus and B. suis were designed and purchased from Sigma-Genosys (The Woodland, TX). Oligonucleotides were suspended in 3× SSC (Ambion) at a final concentration of 40 μM before robotic arrayed in triplicate onto ultraGAPS

coated glass slides (Corning) using a Spotarray 72 microarray printer (Perkin Elmer, Downer’s Grove, ILL). Printed slides were steamed, UV cross-linked and stored in desiccators until use. Sample preparation and slide hybridization Labeling and hybridization procedures were adapted from a protocol developed by The Institute for Genomic Research [67]. Briefly, 10 μg of B. melitensis 16 M total RNA were reverse-transcribed overnight using 6 mg of random hexamer primers (Invitrogen), 0.6 μl 50× dNTPs (Invitrogen)/aa-dUTP (Ambion) mix (2:3 aa-dUTP:dTTP) and 400 U Superscript III (Invitrogen). The reaction was stopped by incubating the samples with 1 M NaOH at 65°C for 15 min and neutralized by subsequently adding 1 M HCl. Unincorparated aa-dUTPs and free amines were removed by column passage (Qiagen PCR Purification Kit, Quiagen). Speedvac-dried samples were rehydrated in 0.1 M Na2CO3 buffer (pH 9.0) and labeled with Cy3-ester (Amersham Pharmacia Biosciences). After one hour incubation in the dark, uncoupled dye was removed by column filtration (Qiagen) and Cy3 incorporation calculated using the NanoDrop® ND-1000 (NanoDrop).