title: NADC_Bordetella_bronchiseptica_v1.0
status: Public on Jan 09 2007
contact.city: Ames
contact.first: Tracy
contact.middle: L
contact.last: Nicholson
contact.email: tnicholson@nadc.ars.usda.gov
contact.state: IA
contact.address: 2300 DAyton Ave
contact.department: Respiratory Diseases of Livestock Research Unit
contact.country: USA
contact.institute: National Animal Disease Center
contact.phone: 515-663-7349
organism: Bordetella bronchiseptica RB50
description: Bordetella bronchiseptica 70-mer oligonucleotide array
manufacture_protocol: Using ArrayOligoSelector, one 70-mer  oligonucleotide probe was selected to represent each B. bronchiseptica RB50 ORF. The 70-bp oligonucleotide probes were synthesized (Illumina, San Diego, CA), resuspended in 3XSSC to a final concentration of 60pmol/µL, and spotted onto poly-L-lysine-coated microscope slides, using a BioRobotics Microgrid II microarrayer (Genomic Solutions, Ann Arbor, MI). Each probe was printed three times per slide. Oligonucleotide microarrays were first prehybridized for 1 hour in 5X SSC, 1% BSA and 0.1% SDS at 42oC, followed by washing with H2O and then with isopropanol and dried by centrifugation for 5 minutes at 50xg. Following prehybridization, 45µL hybridization solution (labeled cDNA, 5µg tRNA, 2XSSC, 25% formamide, 0.1% SDS) was applied to oligonucleotide microarrays and incubated in a humidified chamber overnight at 50oC. Arrays were subsequently removed from humidified chambers and quickly submerged and washed in 1XSSC and 0.05% SDS for approximately 2 minutes, followed by two additional washes for 2 minutes each in fresh 0.06XSSC. Slides were then dried by centrifugation for 5 minutes at 50xg, scanned using a GenePix 4000B microarray scanner, and analyzed with GenePix Pro software (Axon Instruments, Union City, CA). Spots were assessed visually to identify those of low quality and arrays were normalized so that the median of ratio across each array was equal to 1.0. Automatically and manually flagged spots, spots with the sum of medians (635/532) signal intensity less than or equal to 100, and spots with signal intensity below threshold (sum of median intensities plus one standard deviation above the mean background) were filtered out prior to analysis. Ratio data from the three biological replicates were compiled and normalized based on the total Cy3% intensity and Cy5% intensity to eliminate slide to slide variation.