Two-color ratio-coding of chromosome targets in fluorescence in situ hybridization: quantitative analysis and...

Ratio coding in fluorescence in situ hybridizations has the potential to identify more DNA and RNA targets simultaneously using fewer fluorescent labels than other multi-color techniques. Ratio coding uses hybridization probes containing different proportions of two or more distinguishable labels to stain each target. In order to better define the limits of ratio coding applied to chromosome detection, we have 1) examined methods of processing electronic images of ratio-coded hybridizations to increase our ability to visually and quantitatively distinguish different stained targets and 2) examined the reproducibility of target identification. A number of hybridizations were performed using eight 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 interphase nuclei. Greater visual discrimination was afforded by color composite images which expanded the color range across the entire visual spectrum. Quantitative ratio measurements on 25 metaphase spreads predicted that eight different chromosomes can be identified simultaneously with 96% accuracy using whole chromosome probes. Analysis of a smaller data set predicted that six different chromosomes can be identified with 97% accuracy using repetitive sequence probes.