Prehybridization of the array slides was performed for 4h in filtered prehybridization solution [25% formamide, 5x SSC, 10mg l-1 BSA (fraction V), 0.1% SDS] at 42℃. Slides were briefly washed in ethanol and dried by centrifugation at 1000g for 5 min. Hybridization of the probe was performed using hybridization solution (25% formamide, 5x SSC, 0.1% SDS, 0.1µg poly(A) ml-1, 1x Denhardt's solution and 80pmol Cy3 and Cy5 combined probe). The hybridization solution containing the Cy-Dye-labelled cDNA was heated to 95℃ for 3 min, Hybridization is performed by dvalytix hybridaization macine.
Scan protocol
After hybridization, slides were washed in wash buffer I (2x SSC/0.1% SDS) for 2min at 42℃, in wash buffer II (0.2x SSC) for 2min at room temperature and then twice in wash buffer III (0.05x SSC) for 2min at room temperature. All washes were done with vigorous shaking of the microarray slides. The slides were dried by centrifugation at 1000g for 5min, and then analysed using an Fuji FLA-8000 scanner and Array gauge ver.2.0 software (Fuji film)
Description
RNA was isolated from W3110 cells, grown in LB media.
Data processing
Normalization is performed by TREBAX, using MA-plots. MA-plots can reveal the spot artifacts globally and show the intensity-dependent logarithmic ratio of raw microarray data. In MA-plots, we calculate two parameters, average of logarithmic transferred intensity As=(log(Ts)+log(Rs))/2 and logarithmic ratio of intensity Ms=log(Ts/Rs) Here, Ts and Rs are the intensity of target and control experiments for sth spot, respectively.By plotting values of As on the abscissa and Ms on the ordinate of a coordinate system, it is possible to evaluate the bias error with respects to the average logarithmic intensities. Normalized log ratio M’’s is estimated as the difference between Ms and baseline M’s. Here, using a relation between Ms and As, (Ms=f(As)+es, es is the difference between Ms and f(As) for gene s) by MA plot; the baseline for sth spot is estimated by M’=f(A). The genes whose signal intensity is regarded as zero are eliminated in the present analysis. With this methodology, it is assumed that there is no large error due to expression intensity in the majority of the spots and that expression change does not occur on the majority of the spots.
