title: Wheat custom oligonucleotide array for stripe rust study
status: Public on May 01 2009
contact.city: Raleigh
contact.first: Tristan
contact.last: Coram
contact.email: tristan.coram@ars.usda.gov
contact.state: NC
contact.address: 4118 Williams Hall, NC State University
contact.country: USA
contact.institute: USDA-ARS, NC State University
organism: Triticum aestivum
manufacture_protocol: Probes were selected for construction of the custom microarray based on their identification as being differentially expressed in previous studies of both Yr5-mediated all-stage resistance and Yr39 high-temperature adult-plant resistance to stripe rust in wheat. These previous studies both used the commercial Affymetrix Wheat GeneChip (Affymetrix, Santa Clara, CA) and identified a total of 343 probes as differentially expressed that could be used in the present study. The unigenes for each of the 343 differentially expressed probes were identified and DNA sequences extracted using HarvEST (Affymetrix Wheat1 Chip version 1.52). Custom 60-mer oligonucleotide probes were designed to each sequence using the publicly available eArray software (Agilent Technologies, Santa Clara, CA). Oligonucleotide probes for six positive housekeeping controls were also designed (Elongation factor 1-alpha, Sucrose synthase, Ubiquitin, CYP86, Actin and GAPDH), as well as antisense-orientated probes for these six to represent negative controls. A biotin-labeled oligonucleotide was included as a hybridization control, and blank-buffer spots were used as background control. In total, there were 385 features on the microarray
  The 60-mer oligonucelotide probes were re-suspended at a final concentration of 60 µM in 1X ArrayIt® Micro Spotting solution (TeleChem International, Sunnyvale, CA). All features were arrayed onto 4-well (11-mm) epoxy-coated masked slides (Tekdon, Myakka City, FL) such that each feature was printed twice within each of the four wells of each slide. Slides were printed using a piezo electric dispensing Scienion sciFlexarrayer S3 (BioDot, Irvine, CA). Slides were pre-hybridized in filter-sterilized and pre-warmed (42 ºC) 1% bovine serum albumin (BSA), 15X SSC for 10 min with agitation (80 rpm) at room temperature in a Coplin jar, followed by dip rinsing 20 times in sterile water (repeated 5 times in fresh water) and spinning dry in a slide mini-centrifuge for 15 seconds.