Two-color microarrays used for gene expression profiling are hybridized with cDNA prepared from two samples to be compared and that are labeled with two different fluorophores.
Fluorescent dyes include Cy3, which has a fluorescence emission wavelength of 570 nm (green part of the light spectrum), and Cy5 with a fluorescence emission wavelength of 670 nm (red part of the light spectrum). The two Cy-labeled cDNA samples are mixed and hybridized to a single microarray that is then scanned in a microarray scanner to visualize fluorescence of the two fluorophores after excitation with a laser beam of a defined wavelength.
Relative intensities of each fluorophore is then used in ratio-based analysis to identify up-regulated and down-regulated genes.
These one-color microarrays are designed to give estimations of the absolute levels of gene expression. Therefore the comparison of two conditions requires two separate single-dye hybridizations.
As only a single dye is used, the data collected represent absolute values of gene expression. These may be compared to other genes within a sample or to reference "normalizing" probes used to calibrate data across the entire array and across multiple arrays.