Intensity-based analysis of two-colour microarrays enables efficient and flexible hybridization designs. Nucleic Acids Res

In two-colour microarrays, the ratio of signal inten-sities of two co-hybridized samples is used as a relative measure of gene expression. Ratio-based analysis becomes complicated and inef®cient in multi-class comparisons. We therefore investigated the validity of an intensity-based analysis proced-ure. To this end, two different cRNA targets were hybridized together, separately, with a common reference and in a self±self fashion on spotted 65mer oligonucleotide microarrays. We found that the signal intensity of the cRNA targets was not in¯uenced by the presence of a target labelled in the opposite colour. This indicates that targets do not compete for binding sites on the array, which is essential for intensity-based analysis. It is demon-strated that, for good-quality arrays, the correlation of signal intensity measurements between the differ-ent hybridization designs is high (R> 0.9). Furthermore, ratio calculations from ratio- and intensity-based analyses correlated well (R> 0.8). Based on these results, we advocate the use of separate intensities rather than ratios in the analysis of two-colour long-oligonucleotide microarrays. Intensity-based analysis makes microarray experi-ments more ef®cient and more ¯exible: It allows for direct comparisons between all hybridized samples, while circumventing the need for a reference sample that occupies half of the hybridization capacity