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 two novel assays designed to screen for compounds that affect the expression of transcription factors and regulated genes involved in inflammation. Small changes in expression for any of the genes will be quantified for cells exposed to putative drug candidates. One assay will quantify two specific transcription factors involved in inflammation and several genes they regulate (16 total). Another will test expression of a wide panel of transcription factors implicated in the immune response alongside factors controlling other physiological pathways (300 total). A model library of 2000 novel steroidal compounds will be screened. ArrayPlateTM allows DNA array technology to be used in high throughput - with accuracy and precision. Cells are lysed in the presence of nuclease protection oligonucleotides, which hybridize to and protect all targeted mRNAs. After nuclease digestion oligonucleotides bound stochiometrically to targeted sequences are quantified on gene chips. Hybridization specificity is confirmed within each sample. The method is sensitive enough to measure very rare mRNAs - down to one mRNA molecule per cell, from 30,000 cells grown in a 96-well plate.
