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Background  Quantitative real-time PCR (qPCR) has been the method of choice for the quantification of mRNA. Due to the various artifactual factors that may affect the accuracy of qPCR, internal reference genes are most often used to normalize qPCR data. Recently, many studies have employed computer programs such as  GeNorm ,  BestKeeper  and  NormFinder  in selecting reference genes, but very few statistically validate the outcomes of these programs. Thus, in this study, we selected reference genes for qPCR of liver and ovary samples of yellow (juvenile), migratory (silver) and 11-KT treated juveniles of New Zealand shortfinned eels ( Anguilla australis ) using the three computer programs and validate the selected genes statistically using  REST 2009  software and the Mann-Whitney test. We also tested for the repeatability of use for the best reference genes by applying them to a data set obtained in a similar experiment conducted the previous year.    Results  Out of six candidate genes, the combination of  18 s  and  eef1  was found to be the best statistically validated reference for liver, while in ovary it was  l36 . However, discrepancies in gene rankings were found between the different programs. Also, statistical validation procedures showed that several genes put forward as being the best by the programs were in fact, regulated, making them unsuitable as reference genes. Additionally,  eef1  which was found to be a suitable - though not the top ranked - reference gene for liver tissues in one year, was regulated in another.    Conclusions  Our study highlights the need for external validations of reference gene selections made by computer programs. Researchers need to be vigilant in validating and reporting the rationale for the use of reference gene in published studies.

The use of reference gene selection programs to study the silvering transformation in a freshwater eel Anguilla australis: a cautionary tale