title: American and European eel microarray
status: Public on Dec 01 2014
contact.city: Arcachon
contact.first: lucie
contact.last: baillon
contact.address: place du docteur Peyneau
contact.country: France
contact.institute: université de Bordeaux UMR ePOC
organism: Anguilla anguilla
  Anguilla rostrata
manufacture_protocol: One thousand oligonucleotide probes (60mers) were designed from a set of transcripts comprising both a American and European eels transcriptome assembled from a previous  RNA-seq study from hepatic transcriptome of 48 wild eels, collected in 8 sites (4 Canadian and 4 French sites; 6 fish per site) presenting a contamination gradient and a wide range of physicochemical conditions, was determined by high throughput RNA sequencing using Illumina Hiseq2000 technology. A total of 11,547 unique genes of known function were identified (evalue<10-3; size > 200 bp). In parallel, the contamination level of fish in 8 metals and 25 organic pollutants was determined and the temperature, salinity and dissolved oxygen concentration of each sampling sites were measured. Then, we carried out correlation analyses between the transcriptional level of a given gene and (i) contaminant concentrations in each individual fish, (ii) condition indices, (iii) physic-chemical characteristics of sampling sites by means of the factor analysis for multiple testing (FAMT) method (Friguet et al., 2009). The FAMT model was used with a unique threshold of 0.003 in order to identify genes for which their expression levels were more likely correlated with a single natural or anthropogenic factor. In total, 61 genes were significantly correlated with Ag, 64 with As, 60 with Cd, 64 with DDT, 49 with HCB, 55 with Hg, 35 with PBDE-47, 51 with PCB-sum, 68 with trans-nonachlor, 115 with lindane, 51 with hepatosomatic index (HSI), 18 with the Fulton condition factor (K), 68 with spleen-somatic index (SSI), 91 with salinity and 84 with sampling site temperature. To reach a total of 1000 candidate genes 66 genes for which their transcription level was significantly correlated with two factors were retained.  The probes were re-suspended in 3X SSC buffer  and printed on Corning® UltraGAPS TM Coated Slides with the Q-array Mini (Genetix) at the "Génome et transcriptome" (GeT) microarray platform (Toulouse, France). SPM3 (Telechem) Microarray Printing Pins were used to deposit 2nl per spot (100µm/spot) onto the slide.
  The probes were then crosslinked with UV irradiation. The microarray layout consists of a 4-by-4 metagrid with 11 x 18 spots in the subgrids. Each line of 4 subgrids is separated by a line labeled spots in Cy3 and themselves separated by 5 lines of non-labeled spots. Each probe was triplicated on the microarray improving the robustness and repeatability of the measurements. The 1000 probes were deposited in a 4 x 4 metagrid.