DNA microarray methodologies have proven to be an indispensable tool for genome-wide transcriptional profiling of organs, tissues, and cells. Here, we present a protocol for the optimized isolation and preparation of RNA from rat microvessels (including arteries, veins, and lymphatics) for subsequent use in two-color microarray analysis. The investigation of wide-ranging vessel sizes from all three vessel lineages necessitates an RNA isolation strategy that can effectively isolate high-quality RNA from varying and often very small quantities (<1 mg) of fibrous vessel tissue. Additionally, the lack of sample biomass necessitates the use of amplification strategies to generate enough RNA for use in microarray analysis. While the methods presented here were developed for use with two-color microarray analysis, the procedures and general concepts are applicable to most fluorescence-based microarray platforms.