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The plant genes involved in cellular signaling and metabolism have not been fully identified, while the function(s) of many of those which have are as yet incompletely characterized. Gene expression analysis allows the identification of genes and the study of their relationship with cellular processes. There are several options available for studying gene expression, including the use of cDNA and microarray libraries and techniques such as suppression subtractive hybridization (SSH), differential display (DD), RNA fingerprinting by arbitrary primed PCR (RAP), expressed sequence tags (EST), serial analysis of gene expression (SAGE), representational difference analysis (RDA), cDNA-amplified fragment length polymorphism (cDNA-AFLP) and RNA sequencing (RNA-Seq). Focusing on defense-related processes in plants, we present a brief review and examples of each of these methodologies and their advantages and limitations regarding the study of plant gene expression.