A quantitative high-throughput gene chip assay, ArrayPlateTM, based on nuclease protection has been developed for drug discovery. We will use this technology to develop an assay that can measure in a single well of a 96-well microplate, the differentiation of stem cells to osteoblasts or myoblasts as a model system for stem cell differentiation. This assay can be used to screen for compounds that initiate differentiation along a specified pathway. Our patented method applies nuclease protection protocols to gene chips. This allows DNA array technology to be used in high throughput - with accuracy and precision. Briefly, cells are lysed in the presence of nuclease protection oligonucleotides that hybridize to and protect all targeted mRNA sequences. After nuclease digestion, oligonucleotides bound stoichiometrically to targeted sequences are quantified on gene chips, relative to the oligonucleotides as standards. Hybridization specificity is confirmed within each sample. The method is sensitive enough to measure genes expressed at very low levels - down to one mRNA molecule per cell, from 30,000 cells. The cells are grown and treated in individual wells of a 96-well plate.
