A total of forty food-borne fungi obtained from the Agricultural Research Council culture collection (ARC), Pretoria, South Africa, were grown on 1.5% malt extract agar at 25°C for 1-2 weeks. Total genomic fungal DNA was extracted following the DNA extraction protocol described by Raeder and Broda (1985) and column-purified using the QIAquick PCR Purification Kit (QIAGEN).
Label
Cy5
Label protocol
Two micrograms of the total fungal DNA extracted was labelled with Cy5 by using a Cy™Dye Post-labelling Reactive Dye Pack (GE Healthcare, UK). Each labelling reaction contained DNA diluted in 5 μl 0.2 M Na2CO3 (pH9) and 2.5 μl Cy5 mono NHS ester 4000 pmol dye resuspended in 12 μl DMSO. The reactions were incubated at room temperature for 90 minutes in the dark. Free dye was removed with the QIAGEN PCR Purification Kit (QIAGEN, Valencia, CA).
Hybridization protocol
Microarray slides were pre-hybridized for 40 min at 42°C in a solution containing 3.5× SSC, 0.2% SDS, and 1% BSA (Roche Diagnostics). Slides were rinsed three times in deionized water and dried with N2 gas. The Cy5-labeled probe was then dissolved in hybridization solution containing 50% formamide (SIGMA), 25% 2× hybridization buffer (Amersham Pharmacia Biotech), and 25% deionized water. The mixture was denatured at 92°C for 5 min and stored at room temperature for hybridization. The denatured probe (approximately 35 μl) was pipetted directly onto the microarray surface and covered with a glass coverslip (24×60 mm, No.1, Marienfeld, Germany). Slides were placed in a custom-made hybridization chamber (N.B. Engineering Works, Pretoria, South Africa) and incubated for 16–18 h in a 53°C water bath. After hybridization, the slides were washed twice in 2x SSC and 0.2% SDS at 37°C for 6 minutes, once in 0.2x SSC and 0.2% SDS at room temperature for 5 minutes and twice in 0.075x SSC at room temperature for 5 min. The slides were rinsed in deionised water for 2 s and dried by centrifugation at 1000g for 5 minutes.
Scan protocol
Arrays were scanned with an Axon GenePix 4000A scanner and the images were analyzed with GenePix Pro 6.0 software (Axon Instruments).
Description
Sample
Data processing
Raw data processing was performed with GenePix Pro 6.0. Individual net signal intensities were obtained by subtracting the local background from the raw spot intensity value. Irregular spots were manually flagged for removal. Further data analysis was performed in the Microsoft Excel software (Microsoft, Richmond, Washington). Anomalous spots not detected through manual inspection were flagged for removal, if the signal intensity of a spot varied more than 10% from the mean of the sixteen replicates on each slide. Signal intensities of the sixteen replicates were then averaged and intensity values were normalized across slides by global regression on the spot intensity data of the internal transcribed spacer oligonucleotides ITS1, ITS3 and ITS4, which were used as a reference for normalization of all spot intensity data (reference design). The net signal intensity of each spot was divided by the median signal intensity of the sixteen replicates and spots with an SNR ((Signal median – Background median) x Standard deviation Background-1) value below the median were removed from the analysis [32]. Each spot was then either assigned a 1 (present, SNR>/= 2.0) or a 0 (absent, SNR<2.0) according to the median SNR value. The probes with the highest SNR value were considered to be the best target-probe match. These SNR values were then converted to binary score with 1 indicating presence and 0 absence.
