We describe detailed protocols and results with an integrated platform for studying relative transcript expression, including microarray design and fabrication, analysis and calibration algorithms, and high throughput quantitative real-time PCR. This approach optimizes sensitivity and accuracy while controlling the cost of experiments. A high quality cDNA array was fabricated using a restricted number of carefully selected transcripts with each clone printed in triplicate. This focused array facilitated both repeated measurement and replicate experiments. Following normalization and differential expression analysis, we found that experiments with this array identified differentially expressed transcripts with a high degree of accuracy and with high sensitivity to low levels of differential expression. Using a calibration algorithm improved the accuracy of the array in quantifying the relative level of transcript expression. All differentially expressed transcripts identified by the array were independently tested using high throughput quantitative real-time PCR assays. This approach reliably identified transcripts having as low as 1.3-fold differences in transcript expression between RNA samples from treatment- and control groups and was applicable to highly heterogenous tissue sources such as hypothalamus and cerebral cortex.