title: GRASP salmonid 16,006-gene microarray
status: Public on Dec 31 2005
contact.city: Milwaukee
contact.first: Matthew
contact.middle: Lawrence
contact.last: Rise
contact.email: mrise@uwm.edu
contact.state: WI
contact.address: 600 E. Greenfield Ave.
contact.department: Great Lakes WATER Institute
contact.country: USA
contact.institute: University of Wisconsin - Milwaukee
contact.phone: 414-382-1700
organism: Salmo salar
manufacture_protocol: Gene selection
  Microarray fabrication is described in detail in von Schalburg et al. 2005 (BMC Genomics  6:126).  Selection criteria for unique Atlantic salmon (AS) and rainbow trout (RT) cDNAs for inclusion on the 16K microarray were as follows: ESTs (cDNA fragments) were assembled into contiguous sequences (contigs) by PHRAP under stringent clustering parameters (minimum score: 100; repeat stringency: 0.99). Contig consensus sequences and singleton sequences were aligned with non-redundant GenBank nucleotide and amino acid sequence databases using BLASTN and BLASTX, respectively. Threshold for a significant BLAST hit was set at E=1e-15.
  
  It was determined that a cluster must contain at least one "usable" sequence, where "usable" was a) the sequence must be 3' (with high probability; containing polyA signal or having been sequenced with an oligo-dT primer or being at the 3’-end of a contig, with orientation determined by a strong hit against a protein in GenBank’s non-redundant protein database); b) be a sequence stretch containing more than 400 bp and c) the sequence must be at least 95% similar to the consensus of the contig.
  
  It was also determined that if a cluster was a singleton or singleton-equivalent (where all sequences were from the same plate or library thus not providing sufficient evidence for non-chimera status), then the cluster selection was reinforced either by a) a significant BLAST hit, E<1e-15 (BLASTN or BLASTX), or b) it having 94% (or more) identity with a homolog (either paralog or ortholog) covering at least 400 nucleotides. If the cluster was a non-singleton, it was determined that it must be a) one "block" having no regions in the interior of the contig covered by only one sequence (to decrease probability of chimeras) and b) of high enough overall quality (with an overall score > 95% positions without conflicts, weighted by number of sequences which support the consensus) and c) have few leading and trailing singleton positions (no more than 25%), since such positions make it a de facto singleton.
  
  Approximately 3,500 additional sequences were selected with the following criteria: a) no chosen cluster could have 94% or more identity with another chosen cluster (for 400bp) and b) tentative consensus sequences (TC) identified by TIGR (http://www.tigr.org/) could be included. By these criteria, approximately 1000 clones were picked indiscriminately from both normalized AS and RT cDNA libraries, 800 clones were selected from suppression subtracted hybridization libraries, and 700 sequences were added from requests of potential array users. Additionally, 949 non-overlapping sequences (856 AS, 93 RT) from clones included in the preliminary 3,557-gene chip (plus one T cell receptor beta) were selected. Finally, approximately 500 immune-specific genes were also chosen to bring the total number of genes represented on the chip to 16,006. In the 16,006 cDNA features there are 13,421 AS, 2,576 RT, 4 chinook salmon, 3 rainbow smelt and 2 lake whitefish representatives.
  
  Gene Identification
  EST contigs were built using cDNAs on the array as reference and all ESTs currently in the GRASP database. Subsequent to microarray fabrication, the consensus sequences were screened for repeats using a custom salmonid repeat database with RepeatMasker. Masked consensus sequences were compared to GenBank databases.
  
  Microarray fabrication
  Clones were robotically rearrayed from daughter glycerol stock 384-well plates into 96-well plates pre-filled with 7% glycerol in LB + ampicillin, incubated overnight at 37°C, and checked for uniform optical density. Plasmid inserts were PCR amplified in a Tetrad PTC-200 thermocycler (MJ Research) using 1 ul overnight culture, 0.2 mM M13/pUC forward primer (5'-CCCAGTCACGACGTTGTAAAACG-3'), 0.2 mM M13/pUC reverse primer (5'-AGCGGATAACAATTTCACACAGG-3'), 2 mM MgCl2, 10 mM Tris-HCl, 50 mM KCl, 250 mM dNTPs, 1U AmpliTaq (Perkin Elmer), and nuclease-free H2O (Gibco) to 100 ul. PCR conditions were: 2 min for 95°C; then 35 cycles of 95°C for 30 sec, 60°C for 45 sec, 72°C for 3 min; followed by 72°C for 7 min. Five ul of each PCR product were run on a 1% agarose gel to assess yield and quality. PCR products were robotically cleaned (Qiagen) and consolidated into 384-well plates, lyophilized by speed-Vac, and resuspended in 20 ul 3X SSC. Each purified PCR product concentration was determined and diluted to give a final concentration of 400ng/uL.
  
  All cDNAs were printed as single spots on EZ Rays aminosilane slides (Matrix/Apogent Discoveries) with the Biorobotics Microgrid II microarray printer (Genomic Solutions). Microspot™ 10K quill pins (Biorobotics) in a 48 pin tool were used to deposit approximately 0.5 nl (0.2 ng cDNA) per spot onto the slide. The slides were crosslinked in a UV Stratalinker 2400 (Stratagene) at 300 mJ. The resulting microarrays have a 4-by-12 metagrid layout with 19 X19 spot subgrid, each spot having an approximate diameter and pitch of 100 um and 0.20 mm, respectively. A 280 bp GFP (green fluorescent protein) cDNA was amplified from a GFP clone (Clontech) using the primers (5'-GAAACATTCTTGGACACAAATTGG-3') and (5'-GCAGCTGTTACAAACTCAAGAAGG-3') and printed in each subgrid corner to assist in gridding.
  
  Six exogenous genome (Arabidopsis) cDNAs were amplified from the following clones kindly provided by The Arabidopsis Information Resource:  rubisco activase [GenBank:T41667], protochlorophyllide reductase precursor [GenBank:R30630], chlorophyll a/b-binding protein CP29 [GenBank:N65746], PSII oxygen-evolving complex protein 2 [GenBank:H36167], tonoplast intrinsic protein root-specific RB7 [GenBank:AA067532] and ferredoxin (2Fe_2S) precursor [GenBank:W43249]. The Arabidopsis cDNAs were spotted in quadruplicate on each microarray and used for thresholding (determining number of transcripts present). Also, a ubiquitin normalizer serially diluted (50 pg, 5 pg, 500 fg, 50 fg, 5 fg, 0.05 fg and 0.005 fg) was applied to the array. Spot morphology was assessed by visual inspection, SYBR® Green 1 (Molecular Probes) staining or hybridization with labeled non-specific probe. To check clone tracking, 47 high quality sequences were obtained from randomly-selected wells of the cleaned, consolidated 384-well plates used for microarray printing. Each tracked clone had BLAST identifiers matching gene IDs predicted from the re-array spreadsheet, indicating highly accurate clone tracking throughout the process of microarray fabrication.