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


CONCLUSIONS
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.
Hibbeler
Sascha
S
Department of Evolutionary Ecology, Max Planck Institute for Evolutionary Biology, August-Thienemann-Str, 2, 24306 Plön, Germany. hibbeler@mpil-ploen.mpg.de
Scharsack
Joern P
JP
Becker
Sven
S
D000199:Actins / Q000235:genetics; D000818:Animals; D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D005393:Fish Diseases / Q000235:genetics / Q000276:immunology / Q000469:parasitology; D015870:Gene Expression; D005802:Genes, MHC Class II; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012269:Ribosomal Proteins / Q000235:genetics; D023701:Smegmamorpha / Q000235:genetics* / Q000276:immunology; D025801:Ubiquitin / Q000235:genetics
D000199:Actins; D017931:DNA Primers; D018076:DNA, Complementary; D012269:Ribosomal Proteins; D025801:Ubiquitin