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Open Access Highly Accessed Research article

Housekeeping genes for quantitative expression studies in the three-spined stickleback Gasterosteus aculeatus

Sascha Hibbeler1*, Joern P Scharsack1 and Sven Becker2

Author Affiliations

1 Department of Evolutionary Ecology, Max Planck Institute for Evolutionary Biology, August-Thienemann-Str. 2, 24306 Plön, Germany

2 Department of Aquatic Food Webs, NIOO – KNAW Centre for Limnology, Rijksstraatweg 6, 3631 AC Nieuwersluis, The Netherlands

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BMC Molecular Biology 2008, 9:18  doi:10.1186/1471-2199-9-18

Published: 29 January 2008

Abstract

Background

During the last years the quantification of immune response under immunological challenges, e.g. parasitation, has been a major focus of research. In this context, the expression of immune response genes in teleost fish has been surveyed for scientific and commercial purposes. Despite the fact that it was shown in teleostei and other taxa that the gene for beta-actin is not the most stably expressed housekeeping gene (HKG), depending on the tissue and experimental treatment, the gene has been used as a reference gene in such studies. In the three-spined stickleback, Gasterosteus aculeatus, other HKG than the one for beta-actin have not been established so far.

Results

To establish a reliable method for the measurement of immune gene expression in Gasterosteus aculeatus, sequences from the now available genome database and an EST library of the same species were used to select oligonucleotide primers for HKG, in order to perform quantitative reverse-transcription (RT) PCR. The expression stability of ten candidate reference genes was evaluated in three different tissues, and in five parasite treatment groups, using the three algorithms BestKeeper, geNorm and NormFinder. Our results showed that in most of the tissues and treatments HKG that could not be used so far due to unknown sequences, proved to be more stably expressed than the one for beta-actin.

Conclusion

As they were the most stably expressed genes in all tissues examined, we suggest using the genes for the L13a ribosomal binding protein and ubiquitin as alternative or additional reference genes in expression analysis in Gasterosteus aculeatus.