strain: HPA1 extracted molecule: total nucleic acids cell/tissue type: In vitro grown bacterial cell culture
Treatment protocol
N/A
Growth protocol
Bacterial cells cultured overnight on 5% horse blood/Brain Heart Infusion agar plates at 37 oC
Extracted molecule
other
Extraction protocol
Bacterial colonies were isolated and re-suspended in 200 ul TEG buffer (10 mM Tris-Cl pH 8.0, 1 mM EDTA, 2% glucose). 500 ul Guanidine hydrochloride lysis buffer (8 M guanidine hydrochloride, 0.2% sodium sarkinosate) was added to the bacterial suspension which was then mixed by inversion, 200 ul of chloroform was added to the emulsion and centrifuged at 13000 g for 10 minutes. The DNA was recovered from the upper aqueous layer by precipitation with 100 % ethanol and collected by centrifugation. After washing twice with 70 % ethanol and drying, the DNA pellet was re-suspended in TE buffer (10 mM Tris-Cl pH 8.0, 1 mM EDTA) and stored at -20 oC prior to use.
Label
Cy3
Label protocol
The method used to generate randomly amplified DNA targets is a modification of the method published by Bohlander et al and has been described previously (see references 1 and 2 below) Reference 1: Bohlander SK, Espinosa R 3rd, Le Beau MM, Rowley JD, Diaz MO. 1992 A method for the rapid sequence-independent amplification of microdissected chromosomal material. Genomics. 13:1322-4. Reference 2: Burton, J. E., O. J. Oshota, E. North, M. J. Hudson, N. Polyanskaya, J. Brehm, G. Lloyd, and N. J. Silman. 2005. Development of a multi-pathogen oligonucleotide microarray for detection of Bacillus anthracis. Mol Cell Probes 19:349-57
Hybridization protocol
Slides were pre-hybridised in 5 x SSC, 0.1% SDS and 4 x Denhardts solution, washed in sterile, nuclease-free water followed by 100% propan-2-ol then air-dried. Cy3-labelled randomly-amplified target DNAs were denatured at 95 oC for 3 minutes then diluted to a final concentration of 80 μg/ml in 5 x SSC buffer (Sigma-Aldrich), 0.1% SDS, 4 x Denhardts solution at 50oC. 40 μl of hybridisation mix was applied to the microarray slide, which was then incubated in a humidified multi-slide chamber (Genetix Inc.) for 16 hours. The slides were then washed once in each of the following buffers for two minutes: (A) 1 x SSC, 0.2% SDS, 50oC, (B) 0.1 x SSC, 0.2% SDS, 50oC, (C) 0.1 x SSC, 0.2% SDS, 20oC, then centrifuged to dryness at 1000 rpm for 5 minutes
Scan protocol
scanned using an Affymetrix 428 microarray laser scanner at a gain of sixty
Data processing
After image capture files were saved in Tiff format then quantified using the scan analysis software Bluefuse™ (BlueGnome Inc. Cambridge UK), all data were normalised to the global median for each slide then replicate data points fused according to median fluorescent intensity and name. The data were sorted into pathogen relevant groups and each group analysed for statistical significance. Probe intensities were derived for each sample, the log(2) taken and p values calculated which were then sorted from lowest to highest values within each group. Data points for each group were then ranked first by decreasing fold intensity over human DNA control hybridisation values and then by significance (only oligonucleotides with p < 0.2 in each group were considered).
