Identification of reference genes for RT-qPCR data normalization in Gammarus fossarum (Crustacea Amphipoda)

Abstract

Gene expression profiling via RT-qPCR is a robust technique increasingly used in ecotoxicology. Determination and validation of optimal reference genes is a requirement for initiating RT-qPCR experiments. To our best knowledge, this study is the first attempt of identifying a set of reference genes for the freshwater crustacean Gammarus fossarum. Six candidate genes (Actin, TUB, UB, SDH, Clathrin and GAPDH) were tested in order to determine the most stable ones in different stress conditions and to increase the robustness of RT-qPCR data. SDH and Clathrin appeared as the most stable ones. A validation was performed using G. fossarum samples exposed for 15 days to AgNO3, silver nanoparticles (AgNPs) 40 nm and gold nanoparticles (AuNPs) 40 nm. Effects on HSP90 were evaluated and data normalized using Clathrin and SDH. A down-regulation of HSP90 was observed when G. fossarum were exposed to AuNPs 40 nm whereas no effects were observed when G. fossarum were exposed to AgNPs 40 nm. This study highlights the importance of the preliminary determination of suitable reference genes for RT-qPCR experiments. Additionally, this study allowed, for the first time, the determination of a set of valuable genes that can be used in other RT-qPCR studies using G. fossarum as model organism.