Selection and validation of a set of reliable reference genes for quantitative sod gene expression analysis in C. elegans

Abstract

Background: 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.

Results: 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.

Conclusion: 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.

Figure 1

Pairwise variations (V_n/n+1) for all three biological replicate experiments. A large variation between two sequential normalisation factors means that the added gene has a significant effect and should preferably be included for calculation of the normalisation factor. Addition of a 4^th reference gene makes no further improvement of the normalisation factor in each of the three replicate assays.

Figure 2

Normalized sod mRNA expression levels. The relative expression ratios are the average values from 3 replicate cultures. Bars indicate the 95% confidence interval of the mean (non-overlapping intervals denote significant differences at the 0.05 level). This statistic is a conservative criterion of statistical significance. All sod expression levels were normalized using the geometric mean of cdc-42, pmp-3 and Y45F10D.4.