Labelled tumour and reference DNA were combined together with 135 µg human Cot-1 DNA (Invitrogen).
The list of differentially expressed genes are based on a test that the average expression level is up or downregulated, however for as many as 519 of the reported upregulated genes and 542 of the downregulated there is no overlap in gene expression levels between the tumour and reference material.
Briefly, tumour and reference DNAs (300 ng) were labelled with Cy5-dCTP and Cy3-dCTP, respectively.
Labelled tumour and reference samples were pooled and hybridized onto the arrays according to the protocol.
Labelled tumour and reference DNA was mixed with unlabelled Cot-1 DNA, ethanol-precipitated, dried, and dissolved in hybridisation buffer.
Remaining DNA samples (tumour and reference) from 8 randomly selected sample pairs, used in aCGH analyses, were subjected to quantitative PCR.
Briefly, test (tumour) and reference (peripheral blood lymphocytes from a healthy female) DNA was extracted using standard methods and labelled in nick-translation reactions using fluorochrome-conjugated nucleotides, after which DNA fragment lengths between 300 and 2000 bp were obtained.
Briefly, 21.5 μg of tumour and reference RNAs was fluorescently labelled (CyDye, Amersham Biosciences, Buckinghamshire, England) with Cy5 and Cy3, respectively, during cDNA synthesis using an oligo dT) primer (Amersham Biosciences) and Superscript II reverse transcriptase (Gibco BRL Life Technologies, Rockville, MD, USA).
Briefly, six micrograms of digested and purified tumour and reference DNA was labelled with Cy5-dUTP and Cy3-dUTP (Perkin-Elmer, Wellesley, Massachusetts, USA), respectively, in a random priming reaction using BioPrime DNA Labelling System (Invitrogen, Carlsbad, California, USA).
Optimal signal-to-noise ratios were obtained by hybridizing test (tumour) and reference (normal colon) DNA of similar quality, which was determined by giving similar yield in a Bioscore Screening and Amplification kit (ENZO diagnostics Inc., Farmingdale, USA).
