NATIONAL ENVIRONMENTAL RESEARCH INSTITUTE AARHUS UNIVERSITY Write or delete: DATE GENEPEASE From ecotoxicogenomics to a soil ecological model system.

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NATIONAL ENVIRONMENTAL RESEARCH INSTITUTE AARHUS UNIVERSITY Write or delete: DATE GENEPEASE From ecotoxicogenomics to a soil ecological model system

NATIONAL ENVIRONMENTAL RESEARCH INSTITUTE AARHUS UNIVERSITY Jan. 7, 2011 Paul Henning Krogh Lars Henrik Heckmann Jesper Givskov Sørensen GENEPEASE collembolan studies ›WP 2.2 Transcript response to stress in F. candida ›WP2.2.1 Microarrays ›WP2.2.2 temporal qPCR of selected genes at EC 10 and EC 50 effect levels ›WP3 Transcripts in a long-term mesocosm field model system

NATIONAL ENVIRONMENTAL RESEARCH INSTITUTE AARHUS UNIVERSITY Jan. 7, 2011 Paul Henning Krogh Lars Henrik Heckmann Jesper Givskov Sørensen Microarray and qPCR of a collembolan ›iSQ Chip Folsomia candida Collembola VU version 1.0 for gene expression analysis (Dick Roelofs, VU Amsterdam) ›Microarray overview of the genes that are differentially expressed in control and pesticide exposed F. candida ›The temporal expression of selected target genes will be monitored, using real-time quantitative PCR (qPCR), ›Selected target genes should preferably respond to many pesticides

NATIONAL ENVIRONMENTAL RESEARCH INSTITUTE AARHUS UNIVERSITY Jan. 7, 2011 Paul Henning Krogh Lars Henrik Heckmann Jesper Givskov Sørensen Transcriptomic responses in Folsomia candida – potential outcome ›≈ 100 transcripts may be differential in response to the EC 10 and EC 50 ›Upregulation of transcripts encoding for xenobiotic detoxification and biotransformation enzymes (phases I, II, and III) ›General stress response ›Oxidative stress – antioxidant transcripts ›Chitin metabolism ›Suppression of protein translation (allocating energy to detoxification!) ›Immune responses Nota, B., Bosse, M., Ylstra, B., van Straalen, N.M., Roelofs, D., Transcriptomics reveals extensive inducible biotransformation in the soil-dwelling invertebrate Folsomia candida exposed to phenanthrene. BMC Genomics 10:236. Yuan, Y., Ke, X., Chen, F., Krogh, P.H., Ge, F., Decrease in catalase activity of Folsomia candida fed a Bt rice diet. Subm. to Enviromental Pollution.

NATIONAL ENVIRONMENTAL RESEARCH INSTITUTE AARHUS UNIVERSITY Jan. 7, 2011 Paul Henning Krogh Lars Henrik Heckmann Jesper Givskov Sørensen What is a microarray? Microarrays provide a global overview of the genes that are induced/ suppressed in a model organism in a specific biological context

NATIONAL ENVIRONMENTAL RESEARCH INSTITUTE AARHUS UNIVERSITY Jan. 7, 2011 Paul Henning Krogh Lars Henrik Heckmann Jesper Givskov Sørensen How does a microarray work? Exposed F. candida

NATIONAL ENVIRONMENTAL RESEARCH INSTITUTE AARHUS UNIVERSITY Jan. 7, 2011 Paul Henning Krogh Lars Henrik Heckmann Jesper Givskov Sørensen Real-time quantitative PCR (QPCR) To estimate the initial quantity of a specific DNA sequence (target gene)

NATIONAL ENVIRONMENTAL RESEARCH INSTITUTE AARHUS UNIVERSITY Jan. 7, 2011 Paul Henning Krogh Lars Henrik Heckmann Jesper Givskov Sørensen Principles of QPCR

NATIONAL ENVIRONMENTAL RESEARCH INSTITUTE AARHUS UNIVERSITY Jan. 7, 2011 Paul Henning Krogh Lars Henrik Heckmann Jesper Givskov Sørensen Principles of QPCR

NATIONAL ENVIRONMENTAL RESEARCH INSTITUTE AARHUS UNIVERSITY Jan. 7, 2011 Paul Henning Krogh Lars Henrik Heckmann Jesper Givskov Sørensen Principles of QPCR Baseline (threshold) Cycle threshold (Ct) 3 cycles difference between green and blue sample = 2 3 = 8 fold difference

NATIONAL ENVIRONMENTAL RESEARCH INSTITUTE AARHUS UNIVERSITY Jan. 7, 2011 Paul Henning Krogh Lars Henrik Heckmann Jesper Givskov Sørensen Detoxification phases ›In phase I toxic compounds are modified resulting in more reactive ›metabolites (cytochrome P450s) ›phase II reactive metabolites are conjugated by transferases with chemical groups such as glutathione or glucuronic acid ›phase III specialized transporters recognize the conjugates, and expel them out of the cell Nota et al. 2009