Published online 24 February 2004
Nucleic Acids Research, 2004, Vol. 32, No. 4 e41
© 2004 Oxford University Press
Intensity-based analysis of two-colour microarrays enables efficient and flexible hybridization designs
1 Center for Human and Clinical Genetics, 2 Leiden Genome Technology Center and 3 Department of Medical Statistics, Leiden University Medical Center, Wassenaarseweg 72, 2333 AL Leiden, The Netherlands
*To whom correspondence should be addressed. Tel: +31 71 527 6611; Fax: +31 71 527 6075; Email: p.a.c.hoen{at}lumc.nl
In two-colour microarrays, the ratio of signal intensities of two co-hybridized samples is used as a relative measure of gene expression. Ratio-based analysis becomes complicated and inefficient in multi-class comparisons. We therefore investigated the validity of an intensity-based analysis procedure. 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 influenced 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 demonstrated that, for good-quality arrays, the correlation of signal intensity measurements between the different 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 experiments more efficient and more flexible: It allows for direct comparisons between all hybridized samples, while circumventing the need for a reference sample that occupies half of the hybridization capacity.