Why is measuring ratios necessary when using spotted glass microarrays?
Unlike the synthesis of Affymetrix chips, the microarray spotting process produces features with highly variable spot size and morphology. As a result, a number of factors unrelated to transcript abundance, influence the signal intensity of a feature. The co-hybridization of a control sample, in the case of the conventional microarray method a differentially labeled cDNA, is meant to control for many of these, including:

• spot size and morphology
• quality of DNA spotted on the array
• ORF-to-ORF variation in labeled nucleotide frequency
• differences in hybridization efficiency
• uneven hybridization across the array