Understanding the physics of oligonucleotide microarrays: the Affymetrix spike-in data reanalysed

Abstract

The Affymetrix U95 and U133 Latin Square spike-in datasets are reanalysed, together with a dataset from a version of the U95 spike-in experiment without a complex non-specific background. The approach uses a physico-chemical model which includes the effects the specific and non-specific hybridisation and probe folding at the microarray surface, target folding and hybridisation in the bulk RNA target solution, and duplex dissociation during the post-hybridisatoin washing phase. The model predicts a three parameter hyperbolic response function that fits well with fluorescence intensity data from all three datasets. The importance of the various hybridisation and washing effects in determining each of the three parameters is examined, and some guidance is given as to how a practical algorithm for determining specific target concentrations might be developed.