Cell line DNA was prepared using standard methods ie. cell lysis, proteinase K digestion, phenol extraction followed by precipitation (Sambrook and Russell,2001). Tumour samples were ground in a pestle and mortar and extracted as per Cell line DNA.
Label
Cy5
Label protocol
For CGH experiments, labeled DNA was generated using a Bioprime labeling kit (Invitrogen) according to the manufacturer’s instructions, except that 2 ug of DNA was labeled in a 40 ul reaction with 75 uM Cy5-dCTP (or Cy3-dCTP) [NEN, Boston, MA], plus 60 uM dCTP, and 120 uM dGTP, dATP, dTTP and 40units of Klenow large fragment DNA polymerase. Samples were then incubated over night at 37C. The labelling reaction was terminated by the addition of 90mM EDTA, unincorporated nucleotides were removed by the addition of 32 micrograms of COT-1 DNA and 80mM NaOH and heated to 70C for 10 minutes. Samples were clean up and volume reduced by first adding 0.5 x SSPE (5mM Phosphate, pH 7.4, 0.5mM EDTA) to a total volume of 500 microl, this was then filtered through a 0.1 micro m Ultrafree-MC filter column (Millipore) and centrifuged 6500rpm. The volume was then reduced to 3 micro l/2 micro g of labelled sample by filtering through a Microcon YM-30 filtration unit (Millipore) at 13K rpm.
Cell line DNA was prepared using standard methods ie. cell lysis, proteinase K digestion, phenol extraction followed by precipitation (Sambrook and Russell,2001). Tumour samples were ground in a pestle and mortar and extracted as per Cell line DNA.
Label
Cy3
Label protocol
For CGH experiments, labeled DNA was generated using a Bioprime labeling kit (Invitrogen) according to the manufacturer’s instructions, except that 2 ug of DNA was labeled in a 40 ul reaction with 75 uM Cy5-dCTP (or Cy3-dCTP) [NEN, Boston, MA], plus 60 uM dCTP, and 120 uM dGTP, dATP, dTTP and 40units of Klenow large fragment DNA polymerase. Samples were then incubated over night at 37C. The labelling reaction was terminated by the addition of 90mM EDTA, unincorporated nucleotides were removed by the addition of 32 micrograms of COT-1 DNA and 80mM NaOH and heated to 70C for 10 minutes. Samples were clean up and volume reduced by first adding 0.5 x SSPE (5mM Phosphate, pH 7.4, 0.5mM EDTA) to a total volume of 500 microl, this was then filtered through a 0.1 micro m Ultrafree-MC filter column (Millipore) and centrifuged 6500rpm. The volume was then reduced to 3 micro l/2 micro g of labelled sample by filtering through a Microcon YM-30 filtration unit (Millipore) at 13K rpm.
Hybridization protocol
Labelled samples were hybridised in a 50 micro l hybridisation mixture containing 6x SSPE, 12.5mM EDTA (pH 7.4), 0.1% v/v Tween 20 and 50% deionised Formamide. Samples were heated to 70C (CGH) for 2 minutes, cooled to 37C and pipetted on to a microarray slide and covered with a Hybridslip (22x60 mm), placed in the hybridisation chamber with 150 micro l of 0.5 x SSPE pippeped underneath the slide. The slide chamber was sealed and incubated at 65C for 1 hour, and then transferred to 47C incubator for 1-3 days. Slides were rinsed in 4 x SSPE and 10mM EDTA, transferred to a fresh wash solution (4 x SSPE, 10mM EDTA) and gently agitated until the cover slip detached, and then in a third wash solution (4 x SSPE, 10mM EDTA) for 1 minute, all solution were pre warmed to 42C. Slides were then transferred a 42C wash containing 6 x SSPE and 50% formamide for 5 – 15 seconds, and finally washed for 30 seconds each in 2 x SSPE and 10mM EDTA, and 0.1 x SSPE. Finally slides were rinsed in HPLC grade water and excess solution removed with canned air.
Scan protocol
Hybridised microarray slides were scanned with a GenePix 4000B scanner (Axon Instruments), and images visualised by GenePix software (version 3). Slides were scanned with laser power levels at 100% PMT (Photomultiplyer Tube) voltage levels were altered in order to provide an average Cy5:Cy3 hybridisation ratio across the slide of 1:1. Individual fluoresce intensities rations (Cy5:Cy3) for each cDNA were determined by the software, with the hybridisation signal for each clone being calculated as the median fluorescence of pixels within an defined area of array spot. Spots from cDNA clones representing mitochondrial sequences and spots, which didn’t respond, were removed at this point of data analysis. Data was normalised using Lowess Normalisation.
Description
65C for 1 hour, 42C 3 nights, custom made buffer
Data processing
Data were filtered for quality by automated spot flagging and manual inspection in GenePix software and the normalised using a lowess algorithm.
