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Since its origin in the mid-1990s, gene expression profiling by microarray has become a productive and useful tool in basic science and preclinical research. Current dual-color, high-density cDNA oligo arrays contain 60-mer detectors for the whole human genome. With this powerful technology, expression of RNA samples from cell lines or tissue can be assessed, revealing specific gene expression signatures. The technique includes three major steps: (1) isolation and purification of RNA from cells or tissues, (2) labeling of total RNA, and (3) hybridization with Agilent cDNA microarrays. Conveniently, this technique can be performed with as little as 50 ng of purified total RNA; however, it is important to keep in mind that the quality of the RNA template, namely the level of sample degradation and the presence of contaminants that are carried over from the starting material or introduced during RNA isolation, can significantly impact the efficiency of the labeling reaction and the reliability of the hybridization. In this chapter, the details of each step of this technique are explained thoroughly, while highlighting the key issues that can prevent a failed hybridization.