Inbred lines B73 and Mo17, from the same stocks used to create the IBM mapping lines, were kindly supplied by M. Lee. These lines were increased in the field nursery at Clayton, NC. Individual seeds were placed one per 6 cm pot filled with vermiculite, at a density of 36 pots per flat, and grown in the greenhouse without supplemental lighting for ten days to the two-leaf stage. The greenhouse photosynthetically active radiation levels are ~20% less than outdoor radiation. The two lines were then exposed to 4 h of ultraviolet radiation from UV313 bulbs (Q Panel, Westlake, OH, USA) suspended about 30 cm above the plants. The UV dose was measured at plant height using the UV-B sensor on an International Light radiometer Model IL1400A (International Light, Newburyport, MA); the treatment plants received a total UV dose of 86 Jm-2. Control plants were placed under UV313 bulbs that were covered with polyester (MylarD, US Plastics, Lima, OH, USA), which transmits visible light but excludes UV-B. After UV irradiation the UV bulbs were turned off and the plants were allowed to recover for 4 h in the greenhouse. The second and third seedling leaf from each of four plants was harvested and dropped immediately into liquid nitrogen. Pooling assured sufficient RNA and also decreases biological variability. Total RNA was extracted from frozen tissue using Trizol (Invitrogen Co., Carlsbad, CA), as described in the microarray manufacturer’s protocol (http://www.zmdb.iastate.edu/zmdb/microarray/protocols.html#P3). PolyA-plus messenger RNA was prepared from the total RNA using a Qiagen Oligotex kit (Qiagen Corp., Valencia, CA). Poly(A)+ RNA was isolated using Oligotex (Qiagen Inc., Valencia, CA), and 4 mg of poly (A)+ RNA was used for each cDNA synthesis using Superscript II reverse transcriptase (Life Technologies, Carlsbad, CA). cDNA was labeled using 100 mM Cy5-dUTP or Cy3-dUTP (Amersham Pharmacia Biotech, Piscataway, NJ). Excess nucleotides and primers were removed using QIAquick PCR Purification Kit (Qiagen, Valencia, CA). About 95% of the spotted cDNAs showed significant hybridization when leaf mRNA was used for the experiments. The control sample was labeled with Cy5-dUTP fluorescent dye and the UV treatment sample was labeled with Cy3-dUTP (Amersham Pharmacia Biotech, Piscataway, NJ, USA). Two samples, one labeled with each dye, were mixed and then hybridized to a microarray for 15 h at 60°C. The slides were washed in three wash steps: 2x SSC, 0.5% SDS; 0.5x SSC; and 0.05x SSC. The three washes were at room temperature for 5 min each with gentle shaking. The slides were scanned with a GenePix 4000B Scanner (Axon Instruments Inc., Union City, CA). Normalization between the Cy3 and Cy5 fluorescent dye emission channels was achieved by adjusting the levels of both image intensities. Scanalyze (Eisen, http://rana.lbl.gov/EisenSoftware.htm) was used to quantify the image files from the scanner. Keywords = ultraviolet maize corn seedling leaf
Derived by taking the log base 2 of the median of ratios (MRAT). This does not include standardization across all arrays, so these values are not directly comparable to values from other arrays in this experiment.