All A. pleuropneumoniae strains were inoculated into Brain-Heart Infusion (BHI, Difco Laboratories, Detroit, MI) medium supplemented with NAD: either 15 ug/ml in agar or 5 ug/ml in broth. Cultures were grown at 37°C for 16-18 hours before genomic DNA isolation.
Extracted molecule
genomic DNA
Extraction protocol
A. pleuropneumoniae strains were harvested after growth on agar plates for 16-18 h, resuspended in H2O, and treated with lysozyme (Roche, Laval, QC) and RNase A (Qiagen, Mississauga, ON) for 10 min at room temperature. The cell suspensions were then digested with proteinase K (MBI Fermentas, Burlington, ON) for 1 h at 37°C, and complete lysis was obtained by addition of sodium dodecyl sulfate to a final concentration of 0.1% (wt/vol). Genomic DNA, extracted from the cell lysates by two extractions with phenol-chloroform-isoamyl alcohol (25:24:1) and two extractions with chloroform, was precipitated in ethanol.
Label
Cy3
Label protocol
Five ug of fragmented DNA were fluorescently labelled using direct chemical coupling with the Label-IT (Mirus Corp., Madison, WI) cyanine dyes Cy3 and Cy5 as recommended by the manufacturer.
All A. pleuropneumoniae strains were inoculated into Brain-Heart Infusion (BHI, Difco Laboratories, Detroit, MI) medium supplemented with NAD: either 15 ug/ml in agar or 5 ug/ml in broth. Cultures were grown at 37°C for 16-18 hours before genomic DNA isolation.
Extracted molecule
genomic DNA
Extraction protocol
A. pleuropneumoniae strains were harvested after growth on agar plates for 16-18 h, resuspended in H2O, and treated with lysozyme (Roche, Laval, QC) and RNase A (Qiagen, Mississauga, ON) for 10 min at room temperature. The cell suspensions were then digested with proteinase K (MBI Fermentas, Burlington, ON) for 1 h at 37°C, and complete lysis was obtained by addition of sodium dodecyl sulfate to a final concentration of 0.1% (wt/vol). Genomic DNA, extracted from the cell lysates by two extractions with phenol-chloroform-isoamyl alcohol (25:24:1) and two extractions with chloroform, was precipitated in ethanol.
Label
Cy5
Label protocol
Five ug of fragmented DNA were fluorescently labelled using direct chemical coupling with the Label-IT (Mirus Corp., Madison, WI) cyanine dyes Cy3 and Cy5 as recommended by the manufacturer.
Hybridization protocol
Equivalent amounts (2 ug) of labeled tester and control samples were pooled, lyophilized, and then re-suspended in 42 ul of hybridization buffer [1 x DIGEasy hybridization solution (Roche Applied Science); 0.5 ug/ul of Torulla yeast tRNA (Invitrogen); 0.5 ug/ul of salmon sperm genomic DNA (Invitrogen)]. Labelled gDNA was denatured at 65°C for 5 min and applied to the microarray. Hybridizations were performed overnight at 37°C under 22 x 40-mm glass cover slips in a high-humidity chamber. Microarrays were washed 2 x 5 min at 50°C in 1 x SSC with 0.1% SDS, then 2 x 5 min at 50°C in 0.5 x SSC, and 1 x 5 min at 50°C in 0.1 x SSC.
Scan protocol
After hybridization with labelled gDNA, microarray slides of the 15 reference serovars were scanned using a Chipreader laser scanner (BioRad, Mississauga, ON) according to the manufacturer's recommendations.
Description
App_sero2_rep2
Data processing
Spot quantification, signal normalization and data visualization were performed using ArrayPro Analyzer v4.5 (Media Cybernetics, Silver Spring, MD). Net signal intensities were obtained by performing local-ring background subtraction.
