Improvement of condition for hybridization to obtain high sensitivity in oligonucleotide microarray systems

Abstract

The DNA microarray technology is a very useful tool for a comprehensive analysis of the gene expression profile. On the other hand, cost, sensitivity, accuracy, and reproducibility are some of the many problems that must still be solved for this technology to be widely used. The performance of DNA microarray system such as accuracy and reproducibility is mainly defined by the fidelity of probe DNA for the abundance of cellular RNA by or the hybridization efficiency that the labeled target DNA hybridizes to probe DNA. In this study, we manufactured a highly sensitive oligonucleotide microarray system in order to analyze expression profiles comprehensively. In the conventional immobilized hybridization system, oligonucleotide probes which have high sequence specificities could not hybridize to probe DNA uniformly, and they brought nonuniform hybridization depended on fluorescent dyes. So, in order to reduce the ununiformity of hybridization efficiency, we attempted rotated hybridization system using chamber that the hybridization solution can circulate during the reaction. The rotary hybridization reduces uniformity of hybridization efficiency and results in the reduction of the fluorescent dye bias caused by the different amount of hybridized probe between differently labeled DNA. Moreover, by comparing with the expression profiles obtained in quantitative RT-PCR, we showed that the accuracy of the provided data by rotary hybridization was significantly superior to those by immobilized hybridization. We achieved highly accurate and reproducible DNA microarray analysis system using oligonucleotide microarray system.