Microarray List

Expression profiling was conducted with the 60K Rice Whole Genome Microarray. The 60K microarray was designed to represent all of the genes in rice. In total, 60,727 oligomers were designed from gene-specific regions of both japonica and indica subspecies. These include 58,417 from known and predicted genes and 66 randomized DNA oligomers. Among these, 2310 genes were also designed as antisense oligomers. Oligomer sequences were extracted by Qiagen-Operon based on rice genome information from the Beijing Genomics Institute. Oligomers were synthesized and purified by Qiagen-Operon and spotted on SuperAmine slides using the facilities of David Galbraith at the University of Arizona (http://ag.arizona.edu/microarray/deconvolution.html). A set of two slides of the 60K microarray has 64,896 spot addresses. Each slide is formatted with 48 (12x4) blocks composed of spots (4099) were also included for easy scanning alignment. Each oligomer 70 nucleotides long and with an average Tm of 78 °C was printed in each spot address with a diameter of 100 um.

Noncorrelation of signal and background intensities was confirmed by plotting base 2 log background intensity on the x axis and base 2 log intensity subtracted from background intensity on the y axis. Before normalization, the normal distribution and linear relations of Cy3 and Cy5 intensities were tested by qqplot and a linear regression model, respectively, in R statistical language. The spatial effects on the chip during the hybridization process were checked with spatial.func in the sma package. The variance differences between Cy3 and Cy5 intensities within the microarray were tested with the t test under the assumption of both uniform and nonuniform variances. One- and two-way analyses of variance of the signal intensity differences between microarrays were performed. Median pixel intensities were transformed as log ratios with base 2 and then adjusted by block-by-block Lowess normalization for each slide (Yang et al., 2002). To improve the speci~Acity of our statistical hypothesis in low-intensity regions, we adopted the following empirical criteria: a spot was selected if it was not flagged for its morphology, the diameter was larger than 51 pixels, and the inn tensities of both signals were higher than 500. Multivariate statistical tests such as clustering, principal component analysis, and multidimensional scaling were performed with Acuity 3.1 (Axon Instruments).