|Dardick, Christopher - Chris|
|Frank, Bryan C.|
|Yi, Yi Jia|
|Chou, H. H.|
|Lee, Geun Cheol|
Submitted to: PLoS One
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/11/2008
Publication Date: 10/6/2008
Citation: Jung, K., Dardick, C.D., Phetsom, J., Canlas, P., Seo, Y., Shultz, M., Ouyang, S., Yuan, Q., Frank, B., Ly, E., Yi, Y., Hsia, A., An, K., Chou, H., Rocke, D., Lee, G., Schnable, P., An, G., Buell, R., Ronald, P. 2008. Construction and validation of a 45K rice oligonucleotide array. PLoS One. 3(10):e3337. Interpretive Summary: New tools allow researchers to monitor the expression of thousands of genes simultaneously. In this report, we describe the construction of a whole genome, 45,000 oligonucleotide microarray for rice, one of the world’s most important crops. This array was designed to be user friendly and to be provided at a relatively low cost with the hopes of bringing this technology to labs throughout the world. The methods described serve as a template for the creation and validation of whole genome oligoarrays. Step-by-step methods are described for analyzing the data and integrating it with other functional genomics tools and data sets.
Technical Abstract: We have constructed and validated a 43,311 oligonucleotide (oligos, 50-70-mers) gene array (NSF 45K) based on 45,116 gene models from release 3 of The Institute for Genomic Research's rice pseudomolecules. To test the array, we generated expression profiles on light- and dark-grown rice leaf tissue from four rice varieties. We identified 1,000 genes showing at least a 3.2 fold induction in the light within a 0.0001 false discovery rate (FDR). Five methods were developed to validate the biological significance of these genes. First, through a standard analysis of variation (ANOVA) test, we demonstrated that the observed changes in gene expression were due to the treatment and not to the variation in dye, samples, and errors. Second, plant gene ontology (GO) slim data analyzed at three FDRs were used to show that genes related to photosynthesis, photorespiration, or chloroplasts were consistently and significantly affected by light/dark treatments. Third, digital northern data based on the number of expressed sequence tags in 19 tissues were used to assess the biological meaning of the data. Fourth, publicly available microarray data (i.e., NCBI GEO, http://www.ncbi.nlm.nih.gov/geo/) were used to verify and refine the candidate gene list. Fifth, we showed that 52 of the candidate genes encode enzymes catalyzing one of the eight steps in the photorespiration pathway. Unlike other publicly available rice array platforms, the rice NSF 45K array contains 4,500 probes designed to detect the alternatively spliced transcripts of 2,096 genes (loci). Through transcriptome analysis, we effectively differentiated the expression patterns and levels of these splicing isoforms.