Test and reference DNA samples (50μl each) were combined, precipitated together with 100μg of human Cot1 DNA (1μg/μl, Invitrogen) and resuspended in 100μl of hybridization buffer (50% formamide, 10% dextran sulphate, 2X SSC, 2% SDS, 4μg/μl yeast tRNA). The samples were denatured at 70oC for 15mins and pre-hybridized at 37oC for 60mins. Meanwhile, array slides were denatured at 95oC for 2mins, allowed to cool and pre-hybridized in blocking buffer (25% formamide, 0.1% SDS, 5X SSC, 1g BSA) at 42oC for 60mins. The array slides were gently washed in 0.1X SSC for 30secs and spun dry at 2000rpm for 3mins. The pre-hybridized test and reference DNA samples were applied onto cover slips and hybridized onto the array slides. The slides were then transferred into a small hybridization chamber containing Whatmann 3MM paper saturated with dH20 and incubated in a rocking hybridization oven at 42oC for 24hrs. Post hybridization, slides were washed in 2X SSC, 0.1% SDS at 45oC for 15mins, then 2X SSC, 50% formamide at 45oC for 15mins, then 2X SSC, 0.1% SDS at 45oC for 30mins and finally followed by 0.2X SSC for 15mins at room temperature. Slides were spun dry in a centrifuge for 3mins at 2000rpm and stored in a dark dry container until scanning.
Scan protocol
Hybridized microarrays were scanned using an Axon GenePix 4000B scanner at 5micron scans for both channels.
Description
None
Data processing
Images were put through a data extraction program called BlueFuse (Blue Gnome, Cambridge UK) along with a clone position file. Primary data analysis and normalisation were carried out using web-based analysis tool called LIMS (Laboratory Information Management System) developed in our laboratory. The microarray data were normalised using lowess and MANOR algorithms and data points smoothed using a 1Mb window.
