The regulation of myocardial gene expression is highly sensitive to any extracellular or intracellular stimulus that affects contractile function. Subtractive suppression hybridization represents a large-scale, unbiased method for detecting transcriptionally and posttranscriptionally regulated genes, both known and unknown, independently of the prevalence of these transcripts. The strength of subtractive hybridization relies on its unbiased nature and its power to extract even low-abundance transcripts. Therefore, the subtraction experiments can reveal "unexpected" gene profiles and can represent a starting point for the cloning and characterization of novel genes. However, the procedure and the subsequent sequencing of the subtracted genes are labor intensive, and, because the method is purely qualitative, it also requires alternative methods of validation.