Intensity-based analysis of two-colour microarrays enables efficient and flexible hybridization designs.
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.
't Hoen
Peter A C
PA
Center for Human and Clinical Genetics, Leiden University Medical Center, The Netherlands. p.a.c.hoen@lumc.nl
Turk
Rolf
R
Boer
Judith M
JM
Sterrenburg
Ellen
E
de Menezes
Renée X
RX
van Ommen
Gert-Jan B
GJ
den Dunnen
Johan T
JT
D000818:Animals; D002232:Carbocyanines; D003116:Color; D051379:Mice; D009693:Nucleic Acid Hybridization; D020411:Oligonucleotide Array Sequence Analysis / Q000295:instrumentation / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013698:Templates, Genetic
D002232:Carbocyanines; C089187:cyanine dye 3; C085321:cyanine dye 5