Disease profiling arrays: reverse format cDNA arrays complimentary to microarrays.

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The identification of differentially expressed genes enables us to understand the molecular mechanisms associated with disease, conditions of stress, drug treatments and developmental processes. Microarrays provide a powerful tool for studying these complex phenomena. Verification of differentially expressed genes and correlation with biological function, which is usually done by northern blot analysis, RNase protection assay or RT-PCR, is the bottleneck in all these protocols. We developed a new type of cDNA array for high-throughput expression profiling of multiple tissues and blood samples (i) for confirmation analysis of statistically significant number of clinical samples (ii) from limited amount of starting material, and (iii) with detailed clinical data from each individual. In contrast to traditional cDNA arrays, these arrays are spotted with a complex cDNA representing the entire mRNA message expressed in a given tissue or blood sample. cDNAs for these arrays were generated using SMART technology and accurately represent the original mRNA population, producing specific and quantitative signals during hybridization. cDNAs on Disease Profiling Arrays were derived from total RNAs of diseased and normal tissues or different blood fractions of patients. These cDNAs were spotted onto nylon membranes along with positive and negative controls. The arrays were then hybridized with gene-specific probes. Hybridization results revealed disease-related as well as patient-specific gene expression patterns between different disease types for these genes. These studies demonstrate that Disease Profiling Arrays are suitable for high-throughput studies comparing the relative abundance of a target gene, for simultaneously detecting differentially expressed genes in a wide variety of tissues and blood samples, and can be used down-stream from cDNA microarrays for confirmation analysis.