title: PulseChip 1
status: Public on Aug 01 2006
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: Cicer arietinum
  Lathyrus sativus
  Lens culinaris
manufacture_protocol: Coram TE and Pang ECK. 2006. Expression profiling of chickpea genes differentially regulated during a resistance response to Ascochyta rabiei. Published in Plant Biotechnology Journal.
  
  A total of 715 ESTs, 41 RGA DNA sequences, and 12 controls were used in the construction of 768-feature microarrays according to minimum information about a microarray experiment guidelines (MIAME). Of the 715 ESTs, 516 were obtained from a previously synthesized and characterized EST collection constructed from cDNA of the ascochyta blight resistant ICC3996 chickpea genotype. Briefly, 1021 ESTs were generated from stem/leaf tissue of ICC3996 harvested after inoculation with A. rabiei spores, and assembled into 516 unigenes employed in the construction of this microarray. A single clone was selected to represent each contig. A further 43 chickpea cDNAs whose sequencing reactions had previously failed were also included in this microarray. The remaining 156 ESTs represent potential defence-related ESTs from a grasspea (Lathyrus sativus) cDNA library constructed from the Mycosphaerella pinodes resistant genotype ATC80878. Finally, the 41 RGA sequences were isolated from lentil (Lens culinaris) genotypes ILL6002 (A. lentis susceptible) and ILL7537 (A. lentis resistant). The 12 controls included negative, printing, and blank buffer controls. All samples were dried in 384-well plates before being resuspended in 10 µL 50% (v/v) dimethylsulphoxide:water at 250 ng/µL, and arrayed onto amino-silanized slides (Corning Incorporated Life Sciences, Acton, MA) using a BioRobotics® MicroGrid II Compact (Genomic Solutions, Ann Arbor, MI) with four Microspot 2500 pins (Genomic Solutions, Ann Arbor, MI). Blank buffer spots, digested vector and PCR primer were also incorporated as negative controls, as well as Cy5-10T as a printing control. Each element was deposited six times with a volume of approximately 6 nL and diameter of 200 µm. Slides were pre-hybridised by blocking in 5X SSC, 0.1% SDS, 25% Formamide, 1% BSA for 45 min at 42ºC, rinsed in distilled water and dried.