Microarray Probe Design

The probes consist of 60-mer oligo sequences of the RAP-DB loci with transcripts based on rice full-length cDNA (representative cDNA), transcripts based on EST support, as well as transcripts based on ab initio gene prediction. The array probe composition is as follows: 32,325 probes corresponding to RAP loci with full-length cDNA support (representative cDNA); 6,943 probes corresponding to RAP loci with EST support; 2,612 probes corresponding to gene predicted loci; and control probes including false positives, non-rice sequences etc. Details on probe design are available here.

The probes mapped in IRGSP-1.0 can be downloadable as GFF file.

Microarray Probe Table

Data table header descriptions
FeatureNum	Agilent feature number
Row	Row
Col	Column
ProbeName	Probe name
Sequence	60-mer oligonucleotide Sequence
Locus ID	RAP locus identifier
AccessionNum	Accession number
Description	Description based on annotation

Microarray Analysis Protocol

Microarray analysis is performed using high-quality total RNA extracted from rice tissues and organs. For most samples with high RNA yield, the total amount of RNA used for labeling is 400 ng per reaction. For samples with very low yield, the maximum amount of extracted RNA is used for labeling.

Sample preparation and hybridization are performed according to Agilent’s protocols for one-color or two-color microarray-based gene expression analysis. For one-color analysis, the one-color spike mix is added to the RNA sample and labeling with cyanine 3-CTP is performed using the Agilent Quick Amp Labeling Kit. For two-color analysis, the two-color spike mix is added and labeling is performed using Cy3-CTP and Cy5-CTP. The labeled cRNA (1650 ng Cy3-labeled for one-color or 825 ng each of Cy3-/Cy5-labeled for two-color) is then fragmented and hybridized on the 4x44K microaarray slide using the Agilent Gene Expression Hybridization Kit in the hybridization oven at 65°C for 17 hours.

After hybridization, the array slide is removed from the gasket while immersed in a dish with Wash Buffer 1 at room temperature. The slide is then washed with Wash Buffer 1 at room temperature for 1 minute, and with Wash Buffer 2 at 37°C for another 1 min. The processed microarray is scanned with the Agilent DNA microarray scanner and the expression data is extracted with the Agilent Feature Extraction software.