title: Atlantic halibut 40K Array
status: Public on Feb 10 2008
contact.city: Halifax
contact.first: Jason
contact.last: Williams
contact.email: jason.williams@nrc-cnrc.gc.ca
contact.state: Nova Scotia
contact.address: 1411 Oxford Street
contact.department: Institute for Marine Biosciences
contact.country: Canada
contact.institute: National Research Council
contact.phone: (902)426-5903
organism: Hippoglossus hippoglossus
manufacture_protocol: Oligonucleotide DNA probes were designed from approximately 13000 available Atlantic halibut ESTs.  The majority of the ESTs were derived by sequencing from the 5’ end of clones from 13 different normalized libraries from eight different tissues and five different developmental stages (Douglas et al., 2007). All EST data have been deposited in GenBank (accession numbers EB029285-EB041700 & EB080851-EB080975), and preliminary annotations are available on the Pleurogene website (www.pleurogene.ca).  Additional unique ESTs from other research groups that were present in GenBank were also used.  Oligonucleotide probes wFere designed using ArrayDesigner software (Premierbiosoft, Palo Alto, CA, USA). Probe sequences were selected to have minimal secondary structure, a GC ratio between 40-60%, a melt temperature of 75 ± 5oC and to be 45-55-mer in length. Uniqueness of the selected probes was ensured by BLAST comparison against all of the ESTs and the zebrafish genome.
  Halibut DNA probes were synthesized at the Atlantic Microarray Facility (Moncton, NB, Canada) by the phosphoramidite method at a 40 nmole scale on a Gene Machines PolyPlex 2 oligonucleotide synthesizer (Genomic Soutions, Ann Arbor, MI, USA). Isolated DNA was analyzed by ESIElectrospray ionisation-Mass Spectrometry using an Agilent VL1100MSD mass spectrometer (Agilent Technologies, Inc., Mississauga, ON, Canada). DNA of low quality was re-synthesized. In addition to the halibut probes, 1344 control spots containing buffer only, and 92 control spots containing an oligonucleotide specific to the chlorophyll synthetase G4 from Arabidopsis thaliana (GenBank Acc. U19382) were included.   Solutions (20 M) in sodium phosphate Schott-Nexterion Spot buffer (Schott, Jena, Germany) were prepared in 384-well plates (Genetix X7020)
  and spotted in quadruplicate on epoxide microarray slides (Schott) to give 39936-feature microarrays. An OmniGrid 100 microarrayer (Genomic Solutions) equipped with SMT-S50 silicon print pins (Parallel Synthesis Technology, Santa Clara, CA, USA) was used.
  Spotted microarrays were analyzed by SpotQC (IDT Inc., Coralville, IA, USA) for any defects in spotting that may have occurred. Microarrays were stored at room temperature in vacuum-sealed microarray pouches (Corning Inc., Corning, NY, USA).