On-chip hybridization kinetics for optimization of gene expression experiments.
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.
Khomyakova
Elena
E
IMSTAR, Paris, France.
Livshits
Mikheil A
MA
Steinhauser
Marie-Caroline
MC
Dauphinot
Luce
L
Cohen-Kaminsky
Sylvia
S
Rossier
Jean
J
Soussaline
Françoise
F
Potier
Marie-Claude
MC
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
D015342:DNA Probes; D009692:Nucleic Acid Heteroduplexes