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.

Validation of Reference Genes for Quantitative Real-time PCR in Diaphanosoma Celebensis: For Chemical Exposure Conditions and Different Ages

“…Reference gene validation has been performed in many other aquatic invertebrates, such as cladocera Daphnia magna 13 , amphipod Gammarus fossarum 14 , and bivalves Pecten maximus L. 37 and Ruditapes philippinarum 38 .…”

“…Commonly used reference genes contain the 18S rRNA, β-actin, and GAPDH genes 10,11,12 in crustaceans (mainly large crustaceans). Few studies have validated reference genes in small crustaceans 13,14 . It has been suggested that the expression of reference genes can be modulated by different environmental stresses 15 , indicating that some housekeeping genes may be involved in pathways other than cellular homeostasis 16 .…”

The anti-lipidemic drug simvastatin modulates Epigenetic Biomarkers in the AmphipodGammarus locusta

“…Since all the housekeeping genes tested (actin-C, eif2, rpl13, gapdh, clathrin) presented the same pattern for all SIM tested concentrations ( Figure 3), it is plausible to hypothesize that G. locusta, at the highest SIM concentration (8 µg.L -1 ), was experiencing an increase in the overall metabolism. A recent study calculated the stability coefficient of several candidate reference genes in Gammarus fossarum using five different algorithms and showed that clathrin and gapdh were the two most stable genes in this species [67]. Given that the reference genes must show a stable transcriptional pattern across treatments [68], the qRT-PCR data was normalized here with clathrin and gaphd, the genes with the most similar transcription levels to the genes of interest.…”

Reference gene selection in multiple Passiflora edulis tissues and under cold stress for RT- qPCR analysis

“…Nonetheless, using RT-qPCR to investigate gene expression levels requires stably expressed reference genes for data normalization, and the expression stability of reference genes directly affects the accuracy of relative quantitative analyses [7,[9][10][11][12]. In theory, reference genes should have stable expression patterns in different samples and under different experimental conditions, yet no reference gene has satis ed these conditions simultaneously [8,11,[13][14][15][16]. To our knowledge, a candidate reference gene may exhibit distinct expression patterns in different tissues within a species, and it may also exhibit various expression patterns under different conditions even in the same tissue [15,17,18].…”