Reference gene validation

A number of reference (housekeeping) genes have been described in the literature and are used at different frequencies. Suzuki and colleagues reviewed 452 papers published in 1999, in six major journals, which used real-time PCR for mRNA quantification (Suzuki et al., 2000) and found that the most frequently used reference gene was GAPDH (33%), followed by b-actin (32%) and 18S rRNA (14%). However, the value of some reference genes has been questioned. For example, GAPDH, a commonly used reference gene, cannot be used in the analysis of breast tissue as its expression is modulated by hormones (Laborda, 1991). The importance of assay- and tissue-specific reference gene validation has also been illustrated by others (Schmittgen and Zakrajsek, 2000, Sabek et al., 2002, Huggett et al., 2005). The choice of a suitable reference gene is dependent the on individual model and requires investigation of the appropriate literature. The choice of reference genes is the main theme of Chapter 4.

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