title: UCD AMDandekar CitrusClusteredComplimentHKfinal_1647_12k oligo array
status: Public on Mar 05 2008
contact.city: Davis
contact.first: Abhaya
contact.middle: M
contact.last: Dandekar
contact.email: amdandekar@ucdavis.edu
contact.state: CA
contact.address: One Shields Ave
contact.department: Plant Sciences
contact.country: USA
contact.institute: University of California, Davis
contact.phone: 530 752 7784
organism: Citrus sinensis
description: Probe sequences are included in the raw data files linked to the related Sample records.  Gene targets without NCBI accessions in the Platform data table were not submitted to a public sequence database. They were derived from assembly citrus04122004 of the citrus EST database at the Core Genomics Facility (CGF), College of Agricultural and Environmental Sciences at UC Davis (http://cgf.ucdavis.edu/). However, the gene targets of this study are not found in the current assembly curated by CGF. In all but a few cases, targets were contigs assembled from one or more ESTs. In these cases, the sequence identifier given in the Platform data table consists of seven digits, beginning with '104' or '105'. (In the CGF database, these identifiers also contained 'CTG' preceding the seven digits.) Target identifiers that are not seven-digit numbers beginning with '104' or '105' were singletons in the assembly. In the Sample raw data files, the sequence identifier given in the Platform data table, the one associated with the probe, appears in the 'Name' column. In the latter, the beginning and ending positions of the oligo are appended with underscores to the identifier. Expression of 28 target sequences on the array were reported in detail in Goudeau et al. 2008 (doi:10.1016/j.plantsci.2007.11.017). References to contigs in the published article use identifiers with 'S' in place of '104' in the seven-digit identifiers used here.
  
  The Platform data table reflects a condensed representation of the array's replicate features.  The full array layout representing all of the individual features (and identifier annotations eg, oligo sequence, positional information, etc.) is linked as a supplementary file at the foot of this record.
manufacture_protocol: The array had four elements: genes of interest, spike-ins, negative controls, and housekeeping genes. All sequences were designed to eliminate cross-hybridization potential. In addition, probes for a given sequence were distributed across the array to minimize effects of local binding conditions. Included were housekeeping sequences for GAPDH (glyceraldehyde-3-phosphate dehydrogenase), β-tubulin, and an 18S ribosomal subunit. These were identical to those validated with real time quantitative PCR (RTQPCR). Also included were 16 negative controls, each represented by two probes for a total 32 features. These were chosen by Combimatrix based on the absence of sequence homologs in plants. 18 additional non-plant genes were selected by Combimatrix as positive “spike-in” controls. Spiked RNA was included in the hybridization reaction, with three genes each at six distinct concentrations. Each positive control was represented by ~80 probes for a total 1406 spike-in features. This large pool of spike-ins provided a strong normalization tool and identified featureless regions on the chip (given the relatively low number of genes of interest) which interfere with hybridization.
  The experimental portion of the array contained 366 genes of interest, each represented by 15 sense and 15 antisense probes, for a total of 10,567 features.
  Using CustomArray 12k (oligonucleotide), probes were synthesized directly on the chip surface, composed of a proprietary porous reaction layer. 12k arrays consist of 12,000 user definable oligonucleotide features, 35-40 nt optimum. Under each probe, a semiconductor electrode used electrochemical detritylation to control sequence synthesis at that spot without a physical mask as in lithographic synthesis. The process yields a 44 micron feature with 1.5 picomole sensitivity. The arrays were designed for one-color biotinilated hybridization experiments.