title: Arborea spruce 32K
status: Public on Dec 20 2011
contact.city: Québec
contact.first: John
contact.last: MacKay
contact.email: john.mackay@sbf.ulaval.ca
contact.state: Québec
contact.address: 1030, avenue de la Médecine, Pavillon C.-E. Marchand
contact.department: Centre d'étude de la forêt
contact.country: Canada
contact.institute: Université Laval
organism: Picea glauca
  Picea sitchensis
manufacture_protocol: The sequences included in the microarray were selected on the basis of reproducible sequence quality form all of the ESTs and FL-cDNA described for P. glauca in Rigault et al. (2011) and P. sitchensis in Ralph et al. (2008) (Table 1A). To obtain a robust probe set, we selected sequences that were either detected in the two species, verified with two technologies (Sanger and 454) or were derived from a FL-cDNA (Table 1A). The probes were 70 nucleotides in length, and were designed to minimize similarity with other sequences in the dataset. Sequence similarity between the probes and known sequences in P. sitchensis and P. glauca was determined from sequence alignments. The microrray contains 25,045 probes that match with known P. glauca sequences; however, they represent 23,853 unique cDNA gene based on the most recent clustering (Rigault et al., 2011), such that 1007 genes are represented by more than one probe. The microarray consists of 33,984 spotted features including 33,024 sample spots (Invitrogen, Carlsbad, California), 240 negative buffer spots, and 480 Spot Report Alien Oligos (Agilent Technologies, Inc., Santa Clara, CA, USA). Oligonucleotides were consolidated into 384-well plates, lyophilized by speed-Vac, and resuspended in 3X SSC to a printing concentration of 30 µM. Oligos were printed on aminosilane slides (Erie, Hudson, NH, USA) with a QArrayMax microarray printer (Genetix Limited, Hampshire, UK) using 946MP2 microarray pins (ArrayIt Corp, Sunnyvale, CA, USA) in a 48-pin tool depositing ~0.5 nL per spot onto the slide. The resulting microarrays had a 4 x 12 subgrid layout with 708 spots per subgrid, each spot having approximate diameter and pitch of 90µm and 160 µm, respectively. A 280-bp GFP (green fluorescent protein) oligonucleotide was printed in subgrid corners to assist in grid alignment during image processing. The slides were crosslinked in a UV Stratalinker 2400 (Agilent Technologies, Inc.) at 300 mJ. Array quality was assessed by visual inspection and hybridization of representative slides from a print run by dye-labelled random 9-mer oligonucleotides. The quality control images were acquired via the GenePix 4200AL scanner (Molecular Devices, CA, USA) at a 10 micron resolution and quantified with the Imagene 8.0 software suite. Printing and quality control was performed by the Vancouver Prostate Centre Microarray Facility, Vancouver, Canada.