2-COLOR RATIO-CODING OF CHROMOSOME TARGETS IN FLUORESCENCE IN-SITU HYBRIDIZATION - QUANTITATIVE-ANALYSIS AND REPRODUCIBILITY

Ratio coding in fluorescence in situ hybridizations has the potential to identify more DNA and RNA targets simultaneously using fewer fluore scent labels than other multi-color techniques. Ratio coding uses hybr idization probes containing different proportions of two or more disti nguishable labels to stain each target. In order to better define the Limits of ratio coding applied to chromosome detection, we have 1) exa mined methods of processing electronic images of ratio-coded hybridiza tions to increase our ability to visually and quantitatively distingui sh different stained targets and 2) examined the reproducibility of ta rget identification. A number of hybridizations were performed using e ight ratios of two fluorescent labels to identify whole chromosomes in metaphase spreads, and using five ratios of two labels on repetitive sequence probes to identify chromosomes in metaphase spreads and inter phase nuclei. Greater visual discrimination was afforded by color comp osite images which expanded the color range across the entire visual s pectrum. Quantitative ratio measurements on 25 metaphase spreads predi cted that eight different chromosomes can be identified simultaneously with 96% accuracy using whole chromosome probes. Analysis of a smalle r data set predicted that six different chromosomes can be identified with 97% accuracy using repetitive sequence probes. Cytometry 27:314-3 26, 1997. (C) 1997 Wiley-Liss, Inc.