SCREENING ARRAYS TO MONITOR APOPTOSIS GENE EXPRESSION

A quantitative high-throughput gene chip assay, based on nuclease protection, has been developed for drug discovery. The assay permits the expression levels of numerous genes to be measured simultaneously in individual samples. We will use this technology to develop an assay that can measure changes in the expression of pro-apoptotic and anti-apoptotic genes in cells treated with experimental compounds in a single well of a 96-well microplate. This assay, performed in 96-well plates, can be used to screen for novel chemical compounds that induce or inhibit the expression of apoptotic genes. A drug that can induce the expression of pro-apoptotic genes and/or decrease expression of anti-apoptotic genes in cancer cells may prove valuable in the treatment of some types of cancers. Our patented method applies nuclease protection protocols to gene chips. This allows DNA array technology to be used in high throughput format with both sensitivity and reproducibility. Briefly, cells, grown and treated in each well of a 96-well plate, are lysed in the presence of nuclease protection oligonucleotides, which hybridize to and protect all targeted mRNA sequences. After nuclease digestion, oligonucleotides bound stoichiometrically to targeted sequences are quantified on gene chips. The method is sensitive enough to measure genes expressed at very low levels -down to one mRNA molecule per cell in samples of 30,000 cells.