Selection of appropriate reference genes for qPCR in the Chinese mitten crab, Eriocheir sinensis (Decapoda, Varunidae)

The accuracy of qPCR depends on the stability of the reference gene used for data normalization. However, the stably expressed reference genes in the Chinese mitten crab, Eriocheir sinensis, have not been well identified under different experimental conditions. In this study, the stabilities of the expressions of 10 candidate reference genes were evaluated in different developmental stages, tissues, and moulting stages of E. sinensis. Our results indicated that UBE and S27 were the most stable reference genes. To validate the suitability of the reference genes, the EcR (ecdysone receptor) gene was analysed among different moulting stages. The results showed that the expression level of EcR was elevated using the least stable reference gene, GST, compared with using the three most stable reference genes. Taken together, our results indicate that reference genes should be assessed and selected in accordance with the experimental conditions, and more than one reference gene should be selected.