SKY

24-colour chromosome painting was performed by the 'spectral karyotyping' (SKY) technique, essentially as described (Schrock et al., 1996; Roberts et al., 1999).

Briefly, metaphases were hybridised with chromosome paints for all the chromosomes simultaneously, each chromosome being labelled directly or indirectly with a distinct combination of up to five fluorochromes: fluorescein, Spectrum Orange, Texas Red, Cy5 and Cy5.5.

Metaphases were imaged on a fluorescence microscope to show all five fluorochromes simultaneously, and the fluorescence analysed using a spectrometer and CCD camera (Spectracube, Applied Spectral Imaging). DNA was counterstained and imaged separately with DAPI (4,6-diamino-2-phenylindole).

The supplied software analyses the spectrum of each pixel of the image, determines which dyes are present and hence which chromosome. Each pixel of the image is then presented false-coloured in a 'classification colour' to show which chromosome spectrum best matches the spectrum recorded.

Representative karyotypes were assembled as follows: all rearranged chromosomes present in at least two metaphases were listed, and the number of copies of each chromosome was the mode.

FISH - Fluorescence in situ hybridisation

Chromosome copy numbers and translocated fragments were verified in a number of additional metaphases analysed by conventional 2- or 3-colour chromosome painting using separate fluorescent dyes for each chromosome paint.

Individual chromosome paints and centromeric repeat probes (prelabelled, from Oncor), were labelled and hybridised as described (Courtay-Cahen et al 2000). For 3-colour analysis FITC, Spectrum Orange (or Cy3) and Cy5 were used.

CGH - Comparative Genomic Hybridisation

Comparative genomic hybridisation (CGH) was done essentially as described (Kallioniemi et al., 1994; Courjal and Theillet, 1997).

Briefly, normal genomic DNA and cell line DNA were labelled with biotin and digoxigenin respectively by DOP-PCR (Telenius, 1992). Cot-1 DNA (GibcoBRL) was added and the paints hybridised to normal male metaphases (Vysis).

The signals were detected with avidin-TexasRed and FITC-conjugated anti-digoxigenin antibody. The slides were mounted with Vectashield (Vector Labs) containing DAPI.

At least 15 metaphases were captured for each cell line on a Zeiss Axioplan II fluorescent microscope using SmartCaptureVP software and analysed by Quips CGH Karyotyper and Interpreter software (Vysis); or on a Leica DMRB Microscope, captured using a Kappa CF 8/1 DXC CCD camera integrating in 256 grey levels, using Starwise software from IMSTAR (Paris, France).

The threshold set for gains corresponded to a mean hybridisation ratio between tumour and normal of >1.2:1, and for losses of <0.8:1.