title: Treenomix spruce 21.8K cDNA microarray
status: Public on Oct 12 2007
contact.city: Vancouver
contact.first: Joerg
contact.last: Bohlmann
contact.email: bohlmann@msl.ubc.ca
contact.state: BC
contact.address: #301 - 2185 East Mall
contact.department: Michael Smith Laboratories
contact.country: Canada
contact.institute: University of British Columbia
contact.phone: 604-822-0282
organism: Picea
description: Corning UltraGaps
manufacture_protocol: Microarray Fabrication and Quality Control
  Clones were selected from 12 spruce cDNA libraries based on a CAP3 assembly (Huang and Madan, 1999; 95% identity and 40 bp overlap) of 50,770 high-quality 3’ ESTs processed for quality as described in Ralph et al. (2006).  This assembly yielded 22,154 contigs and 13,423 singletons.  For each contig, the EST member having the longest quality- and vector-trimmed (QVT) sequence was chosen as the representative. Only those singleton sequences or contig representatives with a QVT of more than 300 bp were considered further.  To further reduce sequence redundancy, all candidate clones were compared against each other using BLASTN and any clones sharing 95% or greater identity over a length of at least 150 bp were considered redundant.  In these cases, the clone with the longest QVT sequence was chosen as the representative on the array.  Following these procedures a set of 21,843 clones were selected.
  Following in silico selection of candidates, clones were robotically rearrayed from daughter glycerol stock 384-well plates into 96-well plates prefilled with 7% glycerol in LB + ampicillin, incubated overnight at 37°C, and checked for uniform optical density.  Prior to PCR, 5 µL of overnight culture was lysed in 100 µL nuclease-free H2O (Invitrogen) for 10 min at 95°C.  Plasmid inserts were PCR amplified in a GeneAmp PCR System 9700 (Applied Biosystems) by using 2.5 µL of lysed culture, 0.2 µM M13 reverse, 0.2 µM -21M13 forward, 15 mM MgCl2, 100 mM Tris-HCL, 500 mM KCl, 250 µM dNTPs, 9U AmpliTaq (Applied Biosystems), and nuclease-free H2O (Invitrogen) to 100 µL.  PCR conditions were as follows: 5 min at 94°C denaturation; 31 cycles of 30 sec at 94°C, 30 sec at 50°C, and 2 min at 72°C; and 7 min at 72°C.  Three microliters of each PCR product were run on a 1.2% agarose gel (E-Gel® Pre-cast Agarose Electrophoresis System, Invitrogen) to assess quality and yield was determined at 260 nm with a SpectraMax plus (Molecular Device).  Out of 21,843 clones, more than 93% of amplicons consisted of a single product of sufficient intensity and of the expected size.  PCR products were robotically cleaned (Millipore) and consolidated into 384-well plates, lyophilized by speed-Vac, and resuspended in 20 µL 50% DMSO.
  cDNAs were printed on Corning UltraGaps slides with the Chipwriter Pro microarray printer (Bio-Rad).  SMP3 Microspot quill pins (TeleChem) in a 48-pin tool were used to deposit ~7 nL (~0.2 ng cDNA) per spot onto the slide.  The resulting microarrays have a 4 x 12 subgrid layout with 528 spots per subgrid, each spot having approximate diameter and pitch of 100 µM and 200 µM, respectively.   A 280 bp green fluorescent protein (GFP) cDNA was amplified from a GFP-containing plasmid (BD Biosciences) using the primers (5’-AGACAAGTTGGTAATGGTAGCGA-3’) and (5’-ACAGGAAACAGCTATGACCA-3”), and printed five spots per subgrid as orientation markers.  A set of five E. coli cDNAs (fus-A, ilv, inf-C, met-E and rps-A) were amplified from genomic DNA using the E. coli ORFmer PCR primer set (Sigma-Genosys) and spotted 24 times each throughout the array.  A set of five human cDNAs (I.M.A.G.E IDs 5320247, 5785983, 7260066, 7483277 and 8683) were amplified from plasmid DNA using –21M13 forward and M13 reverse primers and spotted 24 times each throughout the array.  Other negative controls included pBluescript II SK(+) vector DNA spotted 24 times (Stratagene), polydeoxyadenylic acid DNA spotted 24 times (Amersham Pharmacia Biotech), 50% DMSO spotted 1,149 times and 1,104 empty spots.  An additional set of three human cDNAs (GenBank IDs BC001473, BC001146 and BC007231) were also amplified from plasmid DNA as described above and spotted five times per subgrid.  Using these templates, in vitro transcribed products (MEGAscript SP6 kit, Ambion) were generated according to manufacturer’s instructions and spiked into our hybridization reactions to serve as positive controls for 1st strand synthesis and slide hybridization.  The slides were crosslinked in a UV Stratalinker 2400 (Stratagene) at 120 mJ.  Spot morphology was assessed on all slides by visual inspection, and a representative slide from each print batch was also evaluated using the Paragon DNA Microarray QC Stain Kit (Invitrogen) according to manufacturer’s instructions.  To check clone tracking, high-quality sequences were obtained from randomly selected re-arrayed glycerol stocks, and more than 91% (165 of 180) of these templates had BLAST identifiers matching gene identifications predicted from the rearray spreadsheet, indicating highly accurate clone tracking throughout the process of microarray fabrication.
  Huang X, Madan A (1999) CAP3: a DNA sequence assembly program.  Genome Res 9: 868-877.
  Ralph SG, Yueh H, Friedmann M, Aeschliman D, Zeznik JA, Nelson CC, Butterfield YSN, Kirkpatrick R, Liu J, Jones SJM, Marra MA, Douglas CJ, Ritland K, Bohlmann J (2006) Conifer defense against insects: microarray gene expression profiling of Sitka spruce (Picea sitchensis) induced by mechanical wounding or feeding by spruce budworms (Choristoneura occidentalis) or white pine weevils (Pissodes strobi) reveals large-scale changes of the host transcriptome.  Plant Cell & Environ 29: 1545-1570.