title: On-chip hybridization kinetics for optimization of gene expression experiments.
abstract: DNA microarray technology is a powerful tool for getting an overview of gene expression in biological samples. Although the successful use of microarray-based expression analysis was demonstrated in a number of applications, the main problem with this approach is the fact that expression levels deduced from hybridization experiments do not necessarily correlate with RNA concentrations. Moreover oligonucleotide probes corresponding to the same gene can give different hybridization signals. Apart from cross-hybridizations and differential splicing, this could be due to secondary structures of probes or targets. In addition, for low-copy genes, hybridization equilibrium may be reached after hybridization times much longer than the one commonly used (overnight, i.e., 15 h). Thus, hybridization signals could depend on kinetic properties of the probe, which may vary between different oligonucleotide probes immobilized on the same microarray. To validate this hypothesis, on-chip hybridization kinetics and duplex thermostability analysis were performed using oligonucleotide microarrays containing 50-mer probes corresponding to 10 mouse genes. We demonstrate that differences in hybridization kinetics between the probes exist and can influence the interpretation of expression data. In addition, we show that using on-chip hybridization kinetics, quantification of targets is feasible using calibration curves.
authors.affiliation: IMSTAR, Paris, France.
authors.forename: Elena
authors.initials: E
authors.lastname: Khomyakova
authors.forename: Mikheil A
authors.initials: MA
authors.lastname: Livshits
authors.forename: Marie-Caroline
authors.initials: MC
authors.lastname: Steinhauser
authors.forename: Luce
authors.initials: L
authors.lastname: Dauphinot
authors.forename: Sylvia
authors.initials: S
authors.lastname: Cohen-Kaminsky
authors.forename: Jean
authors.initials: J
authors.lastname: Rossier
authors.forename: Françoise
authors.initials: F
authors.lastname: Soussaline
authors.forename: Marie-Claude
authors.initials: MC
authors.lastname: Potier
mesh_terms: D000818:Animals; D003198:Computer Simulation; D015342:DNA Probes / Q000378:metabolism; D020869:Gene Expression Profiling / Q000379:methods*; D005786:Gene Expression Regulation; D007700:Kinetics; D051379:Mice; D009692:Nucleic Acid Heteroduplexes; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D013816:Thermodynamics; D044366:Transition Temperature
chemical_list: D015342:DNA Probes; D009692:Nucleic Acid Heteroduplexes