Separated after 24 hours and 72 hours by centrifugation (13,000g 10 min) using 150 mL culture and mixed with RNA later AMBION (RNA stabilization solution).
Growth protocol
The bacteria cultures were incubated at 29ÂșC 60% of relative humidity and sessile and planktonic cells were cultured both in the same bootle and separed after 24 and 72 hours by centrifugation.
Extracted molecule
total RNA
Extraction protocol
We used 100 ml cultures samples (planktonic and sessile). Total RNA was isolated by acid hot phenol extraction as described previously by De Vries et al., (1993).
Label
Cy3
Label protocol
The RNA was differentially labeled with Cy3-dCTP and Cy5-dCTP using a CyScribe first-strand cDNA labeling Kitt (Amersham Biosciences).
Hybridization protocol
Pairs of Cy3- and Cy5-labeled cDNA samples were mixed and hybridized with the array as described by Hedge et al. (2000).
Scan protocol
After the arrays were washed, they were scanned using a pixel size of 10 mm with a Scan Array Lite microarray scanner (Perkin-Elmer, Boston, MA).
Data processing
Spot detection, determination of mean signals and mean local background intensities, image segmentation, and signal quantification were performed for the microarray images using the Array-Pro Analyzer 4.0 software (Media Cybernetics, L.P.). The analysis consisted of three steps: background subtraction and data normalization, analysis of variance, and controlling for the expected number of false positives (Quackenbush 2002). The raw data microarray data were normalized to have comparable signal values between the different microarray slides. After background subtraction, data were quantile-normalized using the q-spline method (Workman et al. 2002), attaining signal intensities with the same distributions between all microarray slides in the red and green channels. The signals were transformed to logarithmic scale (log2), and the resulting signals were examined using M-A plots To determine the significance of the expression differences from planktonic and sessile samples, data were subjected to ANOVA analysis, and a p-value <0.01 was considered as significant. Thus an expected 10% of false positives were accepted. Microarray data were analyzed to assess the statistical significance of expression differences between planktonic and sessile organisms after 24 and 72 hours biofilm maturation stages. The analysis consisted of three steps: background subtraction and data normalization, analysis of variance, and controlling for the expected number of false positives (Quackenbush, 2002).