Nicotiana benthamiana 14k Microarray Expression profiling was conducted with the Nicotiana benthamiana 14k Microarray. The 14k Microarray was designed from 14,308 unigenes clustered from 35,571 ESTs at NCBI (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=unigene). Ten 60-nt long probes were designed from each gene starting 60 bp ahead the stop codon and with shifting 10 bp so 10 probes cover 150 bp in the 3' region of the gene. Among these, the direction of 9,911 was known and 10 probes per gene were designed. The direction of 4,397 genes was not known and designed 10 probes starting 250 bp ahead from the end of the unigene and covered 150 bp downstream. For these unknown genes 10 probes were designed for both sense and anti-sense direction, respectively. In this way, 183,594 60-nt long probes were extracted and duplicated in a chip resulting in 360,312 probes in total. Selection marker genes such as gfp, gus, hyg, bar, and kan are included. The average size of probe is 60-nt long with adjusting its Tm value 75 to 85 °C. The microarray was manufactured at NimbleGen inc (http://www.nimblegen.com/). Random GC probes (40,000) 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