Thermodynamics of RNA/DNA hybridization in high density oligonucleotide microarrays
Abstract
High density oligonucleotide microarrays consist of short DNA fragments bound to a substrate, referred to as probes, which are hybridized to fluorescently labeled RNA targets from a biological sample. Ideally, each immobilized DNA sequence hybridizes with the complementary RNA and thus the intensity of each spot on the surface provides a measure of the abundance of that specific RNA in solution. Here 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 versus 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. For each RNA transcript the intensities are fitted with a single free parameter (an effective temperature), which is roughly constant for all cases analyzed. We identify two possible sources of deviations from the Langmuir isotherms: 1) 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 and 2) Decrease of target concentration due to the hybridization between RNA sequences in solution. Taking both effects into account improves considerably the agreement between the Affymetrix Latin square experiments and the model.Keywords
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