Subtractive hybridization, like serial analysis of gene expression (SAGE), RNA arbitrarily primed polymerase chain reaction (RAP-PCR) and microarrays, is a screening method for differentially expressed genes. At first, poly-A+-RNA is isolated and reverse transcribed into cDNA. With the SMART technology, total RNA can be used. For subtractive hybridization, two adaptors are ligated to the tester cDNA. The tester cDNA is then mixed twice with cDNA of the reference sample. Sequences that are present in equal levels in the tester and in the reference cDNA hybridize to each other and are then removed. In contrast, differentially expressed genes are highly enriched and then amplified by PCR. Because subtractive hybridization is a complex multistep procedure, the results should be controlled at each level and positive controls are recommended. Because all screening methods produce a significant number of false positives, the differential expression has to be confirmed by independent methods.