P. gingivalis microclick here arrays were kindly provided by The Institute for Genomic Research (TIGR) (now The J. Craig Venter Institute). Each microarray consisted of 1907 70-mer oligonucleotides spotted in quadruplicate on a glass slide (CMT-GAPS; Corning, Corning, N.Y.). Detailed array information can be viewed at http://www.tigr.org SRT2104 solubility dmso and http://www.brop.org. A total of four slides were used for each planktonic-biofilm pair, where the cDNAs were labeled with the alternative dye and hybridized to the microarray slides using a dye-swapping design. Slides were prehybridized at 42°C in 5× SSC,
0.1% SDS and 2% bovine serum albumin for 2 h and then briefly rinsed with distilled water and isopropanol. Slides were dried by centrifugation for 3 min at 1,500 × g. The labeled cDNAs hybridization mix was heated to 100°C for 2 min before adding to the DNA microarray. Each array was covered with a coverslip and placed inside a hybridization chamber (Corning Incorporated Life Sciences, Acton, MA). Hybridization selleckchem was carried out in a 42°C water bath for approximately 16 h after which the coverslips were removed and the slides washed in 2× SSC, 0.1% SDS at 42°C. The arrays
were washed at room temperature once with 0.1× SSC, 0.1% SDS for 10 min, four times for 1 min in 0.1× SSC, and then rinsed with distilled water followed by 100% ethanol. The arrays were dried immediately by centrifugation (3 min, 1,000 × g). Image and data analysis The hybridized PI-1840 arrays were scanned using an Agilent G2565AA microarray scanner system (Agilent Technologies, Santa Clara, CA). Imagene 6.0 software (Biodiscovery, Los Angeles, CA) was used for spot finding, signal-background segmentation, and intensity quantification. The intensity of each spot was local background
corrected using GeneSight 4.1 (Biodiscovery) and the resultant data were log transformed such that the mean value for each channel (Cy3 and Cy5) had a log ratio of zero. The signal intensities for each dye swap hybridization were combined and the average log ratios were used for all further analysis. The data were normalized using intensity dependent Lowess normalization [19] per spot and per slide to remove the intensity-dependent deviation in the log2 (ratio) values. Identification of differentially regulated genes was performed using the GeneSight 4.1 confidence analyzer [based on an ANOVA approach of Kerr et al [20]]. This statistical analysis uses replicate spots to estimate an empirical distribution of noise. The constructed noise model is then used to determine the statistical measures for the likelihood of false positives above or below a certain expression ratio. The differentially regulated genes were identified at 99% confidence intervals with a cut-off value of log2 > 0.6 or log2 < -0.6. These values correspond to approximately 1.5 fold up- and down-regulated genes, respectively, a ratio considered biologically relevant [21, 22].