Thermodynamics of RNA/DNA hybridization in high-density oligonucleotide microarrays

We analyze a series of publicly available controlled experiments (Latin square) on Affymetrix high-density oligonucleotide microarrays using a simple physical model of the hybridization process. We plot for each gene the signal intensity vs. the hybridization free energy of RNA/DNA duplexes in solution, for perfect matching and mismatching probes. Both values tend to align on a single master curve in good agreement with Langmuir adsorption theory, provided one takes into account the decrease of the effective target concentration due to target–target hybridization in solution. We give an example of a deviation from the expected thermodynamical behavior for the probe set 1091_at due to annotation problems, i.e., the surface-bound probe is not the exact complement of the target RNA sequence, because of errors present in public databases at the time when the array was designed. We show that the parametrization of the experimental data with RNA/DNA free energy improves the quality of the fits and enhances the stability of the fitting parameters compared to previous studies.