High throughput nano-liter RT-qPCR to classify soil contamination using a soil arthropod.

M.E. de Boer, S.. Berg, M.J.T.N. Timmermans, J.T. den Dunnen, N.M. van Straalen, J. Ellers, D. Roelofs

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    Background: To incorporate genomics data into environmental assessments a mechanistic perspective of interactions between chemicals and induced biological processes needs to be developed. Since chemical compounds with structural similarity often induce comparable biological responses in exposed animals, gene expression signatures can serve as a starting point for the assessment of chemicals and their toxicity, but only when relevant and stable gene panels are available. To design such a panel, we isolated differentially expressed gene fragments from the soil arthropod Folsomia candida, a species often used for ecotoxicological testing. Animals were exposed to two chemically distinct compounds, being a metal (cadmium) and a polycyclic aromatic hydrocarbon (phenanthrene). We investigated the affected molecular responses resulting from either treatment and developed and validated 44 qPCR assays for their responses using a high throughput nano-liter RT-qPCR platform for the analysis of the samples.Results: Suppressive subtractive hybridization (SSH) was used to retrieve stress-related gene fragments. SSH libraries revealed pathways involved in mitochondrial dysfunction and protein degradation for cadmium and biotransformation for phenanthrene to be overrepresented. Amongst a small cluster of SSH-derived cadmium responsive markers were an inflammatory response protein and an endo-glucanase. Conversely, cytochrome P450 family 6 or 9 was specifically induced by phenanthrene. Differential expressions of these candidate biomarkers were also highly significant in the independently generated test sample set. Toxicity levels in different training samples were not reflected by any of the markers' intensity of expressions. Though, a model based on partial least squares differential analysis (PLS-DA) (with RMSEPs between 9 and 22% and R
    Original languageEnglish
    Pages (from-to)11
    JournalBMC Molecular Biology
    Publication statusPublished - 2011


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