Dataset Information

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.

SUBMITTER: Mehennaoui K

PROVIDER: S-EPMC6189083 | biostudies-literature | 2018 Oct

REPOSITORIES: biostudies-literature

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

Mehennaoui Kahina K Legay Sylvain S Serchi Tommaso T Guérold François F Giamberini Laure L Gutleb Arno C AC Cambier Sébastien S

Scientific reports 20181015 1

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 ...[more]

PMID: 30323236

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Project description:BackgroundA critical step in the RT-qPCR workflow for studying gene expression is data normalization, one of the strategies being the use of reference genes. This study aimed to identify and validate a selection of reference genes for relative quantification in Talaromyces versatilis, a relevant industrial filamentous fungus. Beyond T. versatilis, this study also aimed to propose reference genes that are applicable more widely for RT-qPCR data normalization in filamentous fungi.ResultsA selection of stable, potential reference genes was carried out in silico from RNA-seq based transcriptomic data obtained from T. versatilis. A dozen functionally unrelated candidate genes were analysed by RT-qPCR assays over more than 30 relevant culture conditions. By using geNorm, we showed that most of these candidate genes had stable transcript levels in most of the conditions, from growth environments to conidial germination. The overall robustness of these genes was explored further by showing that any combination of 3 of them led to minimal normalization bias. To extend the relevance of the study beyond T. versatilis, we challenged their stability together with sixteen other classically used genes such as β-tubulin or actin, in a representative sample of about 100 RNA-seq datasets. These datasets were obtained from 18 phylogenetically distant filamentous fungi exposed to prevalent experimental conditions. Although this wide analysis demonstrated that each of the chosen genes exhibited sporadic up- or down-regulation, their hierarchical clustering allowed the identification of a promising group of 6 genes, which presented weak expression changes and no tendency to up- or down-regulation over the whole set of conditions. This group included ubcB, sac7, fis1 and sarA genes, as well as TFC1 and UBC6 that were previously validated for their use in S. cerevisiae.ConclusionsWe propose a set of 6 genes that can be used as reference genes in RT-qPCR data normalization in any field of fungal biology. However, we recommend that the uniform transcription of these genes is tested by systematic experimental validation and to use the geometric averaging of at least 3 of the best ones. This will minimize the bias in normalization and will support trustworthy biological conclusions.

Dataset Information

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

Publications

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

Similar Datasets

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