DETECTION OF DIFFERENTIALLY EXPRESSED GENES IN PRIMARY TUMOR-TISSUES USING REPRESENTATIONAL DIFFERENCES ANALYSIS COUPLED TO MICROARRAY HYBRIDIZATION

The identification of differential gene expression between cells is a frequent goal in modern biological research. Here we demonstrate the c oupling of representational difference analysis (RDA) of cDNA with mic roarray analysis of the output for high throughput screening. Two prim ary Ewing's sarcoma tissue samples with different biological behavior in vivo were compared by RDA: one which was metastatic and progressed rapidly; the other localized and successfully treated. A modified RDA protocol that minimizes the necessary starting material was employed. After a reduced number of subtractive rounds, the output of RDA was sh otgun cloned into a plasmid vector. Inserts from individual colonies f rom the subtracted library were amplified with vector-specific primers and arrayed at high density on glass slides. The arrays were then hyb ridized with differentially fluorescently labeled starting amplicons f rom the two tissues and fluorescent signals were measured at each DNA spot. We show that the relative amounts of fluorescent signal correlat e well with the abundance of fragments in the RDA amplicon and in the starting mRNA. In our system, we analyzed 192 products and 173 (90%) w ere appropriately detected as being >2-fold differentially expressed, Fifty unique, differentially expressed clones were identified. Therefo re, the use of RDA essentially provides an enriched library of differe ntially expressed genes, while analysis of this library with microarra ys allows rapid and reproducible screening of thousands of DNA molecul es simultaneously. The coupling of these two techniques in this system resulted in a large pool of differentially expressed genes.