pylori at a multiplicity of infection (MOI) of 20. The infected cells were cultured for additional 16 h after which the media was collected and stored for ELISA and BioPlex analyses and the RNA extracted for microarray and real-time PCR studies. RNA extraction and microarray To extract the RNA from the AGS cells, coculture supernatants were removed by aspiration and 1 ml of TRIZOL (Invitrogen, Carlsbad, CA) was added immediately to each well. RNA was extracted as recommended by the manufacturer and was stored at −80°C until further

use. RNA was dissolved in DNase/RNase-free water, quantified by NanoDrop (Fisher Scientific) and set at a concentration of find more ~1.0 μg/μl. The quality of the RNA was confirmed AZD5582 cell line by Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA). Each experiment was repeated four times. Two hundred ng of RNA were used to make biotinylated cRNA using the Illumina TotalPrep RNA Amplification Kit (Ambion, high throughput screening Austin, TX), and hybridized to the Illumina chips for 14 hours at 58°C. After washing and staining, the arrays were scanned with the BeadArray Reader (Illumina Inc.) and analyzed with the GenomeStudio software (Illumina). Microarray data analysis After subtracting the background, the samples were normalized assuming a similar distribution of transcript abundance in all the samples [37]. The net expression level was obtained

by subtracting the intensity obtained on each treatment (including non-treated cells) from the intensity at 0 h (prior to seeding the cells into the plate). Then, the gene levels on the infected BCKDHB cells were compared against the levels on the non-infected

cells setting the p value for the difference at <0.05. Scatter plots comparing the non-infected cells against each one of the other treatments (AGS + WT, AGS + rocF-, AGS + rocF +) were used to select only those genes with > 3 fold difference (up or down-regulated) as compared with the non-infected cells, and p values less than 0.05 (p < 0.05). In addition, the Log10 of the ratio between the normalized intensity in the infected cells and the normalized intensity in the non-infected cells was determined and used to generate heat maps. Quantitative real-time PCR For real-time PCR (qPCR), total RNA extracts were DNase treated and reverse-transcribed with SuperScriptase III (Invitrogen) with random hexamers. TaqMan pre-designed arrays were used to check the levels of mRNA expression of IL-8, using cyclophilin A as housekeeping gene, and following the vendor’s recommendations (Applied Biosystems, Foster City, CA). The 5 μl reaction was subjected to two minutes at 50°C, 10 minutes at 95°C and finally 40 cycles at 95°C for 15 seconds and 60°C for one minute in a 7900HT real-time PCR machine (Applied Biosystems). The delta Ct’s (ΔCt and ΔΔCt) and fold induction of IL-8 were determined using an internal control as calibrator.