Combining SSH and cDNA microarrays for rapid identification of differentially expressed genes

Comparing patterns of gene expression in cell lines and tissues has importa nt applications in a variety of biological systems. in this study we have e xamined whether the emerging technology of cDNA microarrays will allow a hi gh throughput analysis of expression of cDNA clones generated by suppressio n subtractive hybridization (SSH). A set of cDNA clones including 332 SSH i nserts amplified by PCR was arrayed using robotic printing. The cDNA arrays were hybridized with fluorescent labeled probes prepared from RNA from ER- positive (MCF7 and T47D) and ER-negative (MDA-MB-231 and HBL-100) breast ca ncer cell lines. Ten clones were identified that were over-expressed by at least a factor of five in the ER-positive cell lines. Northern blot analysi s confirmed over-expression of these In cDNAs. Sequence analysis identified four of these clones as cytokeratin 19, GATA-3, CD24 and glutathione-S-tra nsferase mu-3. Of the remaining six cDNA clones, four clones matched EST se quences from two different genes and two clones were novel sequences. Flow cytometry and immunofluorescence confirmed that CD24 protein was over-expre ssed in the ER-positive cell lines. We conclude that SSH and microarray tec hnology can be successfully applied to identify differentially expressed ge nes. This approach allowed the identification of differentially expressed g enes without the need to obtain previously cloned cDNAs.