title: Perch 1000 candidate-gene microarray
status: Public on Sep 17 2013
contact.city: Québec
contact.first: Berenice
contact.last: BOUGAS
contact.email: berenice.bougas.1@ulaval.ca
contact.state: QC
contact.address: 1030 avenue de la médecine, IBIS, Pavillon Marchand
contact.department: Biology
contact.country: Canada
contact.institute: Laval university
organism: Perca flavescens
manufacture_protocol: One thousand oligonucleotide probes (58 to 62bp) were designed from a set of transcripts comprising both a yellow perch transcriptome assembled from a previous 454 experiment focusing on metal contaminated and uncontaminated yellow perch (Pierron et al., 2011) and yellow perch transcripts available in online databases. The probes were designed using the command line version of ProDesign (Feng and Tillier, 2007) and synthesized by Sigma®. ProDesign maximizes probe specificity and ensures that each probe cannot be found among the list of interesting genes, as well as among the list of all the unwanted sequences. 6C amine spacers were added to each probe at the 5’ extremity. All probes were resuspended in SSC 3X and stocked in 384-well plates. In total, 219 gene sequences were chosen from the literature according to their role in key biological pathways, such as lipid and energy metabolism, in fish. The other sequences were chosen from the 454 experiment as follows: 40 genes that presented an interesting SNP and 741 genes from the 1297 unique genes that were annotated from either the swissprot or the nr protein database (minimal e-value < 1.0 e-10 using the blastx program from BLAST 2.2.23+, http://blast.ncbi.nlm.nih.gov). Among these 741 genes, 49 genes were manually chosen because of their important role in global metabolism or response to metal exposure, 196 genes were chosen because their expression levels were significantly correlated with hepatic Cd or Cu concentrations (Pierron et al., 2011), and the 496 remaining genes correspond to genes annotated with an e-value lower than 1.0e-30 and that were significantly different among lakes but not directly correlated with metal concentration. We added four positive control spots containing a mix of all probes and four negative spots that only contained SSC 3X and 6C spacers but no DNA probe. The probes were printed on SuperChip®Poly-L-Lysine slides (Thermo Scientific®) with the Spotarray 24 (PerkinElmer Life Sciences). The Stealth 3 Microarray Printing Pins were used to deposit 0.0125 µL (0.011 µg oligonucleotides) per spot onto the slide. The probes were then crosslinked with UV irradiation. The microarray layout consists of a 4-by-4 metagrid with 9×14 spots in the subgrids. Each probe was duplicated side by side and the 1000 probes were deposited in a 4-by-4 metagrid. Finally, the 4-by-4 metagrid is replicated three times on the chip. In total, the 1000 probes are thus replicated six times on each slide, improving the robustness and repeatability of the measurements.