Selection and validation of a set of reliable reference genes for quantitative sod gene expression analysis in C. elegans
1 Department of Biology and Center for Molecular Phylogeny and Evolution, Ghent University, B-9000 Ghent, Belgium
2 Center for Medical Genetics Ghent, Ghent University Hospital, B-9000 Ghent, Belgium
BMC Molecular Biology 2008, 9:9 doi:10.1186/1471-2199-9-9Published: 22 January 2008
In the nematode Caenorhabditis elegans the conserved Ins/IGF-1 signaling pathway regulates many biological processes including life span, stress response, dauer diapause and metabolism. Detection of differentially expressed genes may contribute to a better understanding of the mechanism by which the Ins/IGF-1 signaling pathway regulates these processes. Appropriate normalization is an essential prerequisite for obtaining accurate and reproducible quantification of gene expression levels. The aim of this study was to establish a reliable set of reference genes for gene expression analysis in C. elegans.
Real-time quantitative PCR was used to evaluate the expression stability of 12 candidate reference genes (act-1, ama-1, cdc-42, csq-1, eif-3.C, mdh-1, gpd-2, pmp-3, tba-1, Y45F10D.4, rgs-6 and unc-16) in wild-type, three Ins/IGF-1 pathway mutants, dauers and L3 stage larvae. After geNorm analysis, cdc-42, pmp-3 and Y45F10D.4 showed the most stable expression pattern and were used to normalize 5 sod expression levels. Significant differences in mRNA levels were observed for sod-1 and sod-3 in daf-2 relative to wild-type animals, whereas in dauers sod-1, sod-3, sod-4 and sod-5 are differentially expressed relative to third stage larvae.
Our findings emphasize the importance of accurate normalization using stably expressed reference genes. The methodology used in this study is generally applicable to reliably quantify gene expression levels in the nematode C. elegans using quantitative PCR.