Computational protocol: Generation of subspecies level-specific microbial diagnostic microarrays using genes amplified from subtractive suppression hybridization as microarray probes

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Protocol publication

[…] A GenePix® 4000B microarray scanner set (Axon instruments, Union City, CA) was used for scanning the microarrays at a resolution of 10 μm. For consistent scanning of all hybridized slides, the laser power and photomultiplier tube (PMT) gain were adjusted to 100%. Scanned image displays were analyzed by quantifying the pixel density (intensity) of each hybridization spot using the GenePix® software program (version 4.0; Axon instruments). Hybridization images presented here are representative images, which were automatically contrast-adjusted by the software. A grid of individual circles defining the location of each DNA spot on the array was superimposed onto the image to indicate each quantified fluorescent spot. Mean signal intensity was automatically determined for each spot. The local background signal was also automatically subtracted from the hybridization signal of each individual spot. The signal-to-noise ratio (SNR) for each spot was calculated based on following formula (): SNR = (signal intensity − background)/SD of background, in which the ‘background’ measurement refers to the local spot background intensity and the ‘SD of background’ was calculated across all pixels, as measured by the GenePix® software. Statistical analysis was performed using Excel 2003 (Microsoft) and Sigmaplot 8.0 (Jandel Scientific, San Rafael, CA). The SNRs from 12 replicate data sets were then averaged to obtain the SNR value of each individual probe. Relative SNRs were obtained by dividing the average SNR value by the mean value of the hybridized template SNR in the same microarray slide. [...] 16S rDNA isolated from each Salmonella strain was amplified by PCR using two universal primers as described by Yoon et al. () and sequenced as described by Yoon et al. (). Multiple alignments were performed using the Clustal X program (). The evolutionary distances were calculated using the Jukes & Cantor method. The phylogenic tree was constructed using a neighbor-joining method () in the MEGA 2 program (). To compare similarities, a 16S rRNA homology matrix was constructed using PairProWin.exe () software, which is based on the Clustal W program. […]

Pipeline specifications

Software tools SigmaPlot, Clustal W, MEGA
Applications Miscellaneous, Phylogenetics