22-μm filter, and stored at −20°C until use. Bacterial strain and growth conditions P. gingivalis strain W83 (kindly supplied by Dr. Koji Nakayama, Nagasaki University Graduate School of Biomedical Sciences) was cultured at 37°C anaerobically (85% N2, 10% H2, and 5% CO2) in

half-strength brain heart see more infusion selleck chemicals (BHI) broth (Becton Dickinson, Sparks, MD) supplemented with 0.5% yeast extract (Difco Laboratories, Detroit, MI), 5 μg/ml of hemin (Sigma), and 1 μg/ml of vitamin K1 (Sigma). RNA isolation and cDNA synthesis Use of high concentrations of antibacterial agents for extended periods of time changes the expression of a large set of genes and the effect may be secondary to the action of the drug [46]. Meanwhile, at sub-lethal concentrations, bacteria may sense antibiotics as extracellular chemicals to trigger different cellular responses such as an altered antibiotic resistance/tolerance profile [47]. Hence, we performed the full-genome gene expression microarrays of P. gingivalis W83 exposed to polyP75 at a concentration of 0.03%, which was previously determined to be MIC against the bacterium [16], for a short period of time. P. gingivalis culture grown to early exponential phase (OD600 = 0.3) was divided in half. One aliquot was left untreated, while the other one was treated with 0.03% polyP75. After incubation of both the bacterial cultures for 2 h under anaerobic

conditions, the bacterial cells were harvested, and total RNA was extracted from the cells using Trizol Reagent (Invitrogen, Carlsbad, CA). RNA quality was monitored by Agilent 2100 Bioanalyzer (Agilent Technologies, OSI-744 research buy Santa Clara, CA), and RNA quantity was measured by spectrophotometer.

All the samples used in this study exhibited A260/A280 ratio of at least 1.8. cDNA was synthesized with 20 μg of total RNA using SuperScript® II Reverse Transcriptase (Invitrogen). Microarray analysis Two individual Cy3-labeled cDNA samples were hybridized into DNA microarrays (Nimblegen Systems, Inc., Madison, WI) containing the whole genome of 1,909 genes of RANTES P. gingivalis W83 for 16 h at 42°C. Five replicates of the genome were included per chip. An average of 19 different 60-mer probes which had at least three mismatches compared to other 60-mers represented each gene in the genome. A quality control check (hybridization) was performed for each array, which contained on-chip control oligonucleotides. Data were extracted from the scanned images using an Axon GenePix 4000B microarray scanner and NimbleScan Version 2.3. Quantile normalization was performed across replicate arrays, and RMA (Robust Multichip Average) analysis was performed to generate gene expression values. Genes evidencing statistically significant changes in expression (>1.5-fold difference) were identified via t-tests (P < 0.05). Assessment of array data quality To confirm the microarray results using qRT-PCR, 10 genes were selected, and specific primers for the selected genes (Table 6) were designed using Primer3 (http://​fokker.