• Rice 3'Tiling Microarray
  • The profiling was conducted with the Rice 3'Tiling Microarray designed from 27,448 genes deposited at IRGSP, RAP1 database (http://rapdb.lab.nig.ac.jp). Among these, 20,507 genes were from representative RAP1 sequences with cDNA/EST supports and 6,941 genes were predicted without cDNA/EST supports. Ten 60-nt long probes were designed from each gene starting 60 bp ahead the end of stop codon and with shifting 10 bp so 10 probes cover 150 bp in the 3' region of the gene. Genes from chloroplast (123) and mitochondria (74) and selection markers such as gfp, gus, hyg, bar, and kan are included. In total, 270,000 probes were designed with the average size of probe is 60-nt long to have its Tm value 75 to 85 °C. The microarray was manufctured at NimbleGen inc (http://www.nimblegen.com/). Random GC probes (38,000) to monitor the hybridization efficiency and four corner fiducial controls (225) were included to assist with overlaying the grid on the image.
  • To assess the reproducibility of the microarray analysis, we repeated the experiment two or three times with independently prepared total RNA. The normal distribution of Cy3 intensities was tested by qqline. The data was nomalized and processed with cubic spline normalization using quantiles to adjust signal variations between chips and Rubust Multi-Chip Analysis (RMA) using a median polish algorithm implemented in NimbleScan (Workman et al., 2002. Genome Biol. 3: research0048.1 - research0048.16 ; Irizarry et al., 2003. Nucleic Acids Research 3:e15).
  • Hybridization
  • Genes on microarray

3 prime tiling microarray