One quadrant of the microarray hybridized to RNA from untreated control ML-1 cells (green fluorochrome) and ML-1 cells four hours after treatment with 20 Gy 137Cs g-rays (red fluorochrome). Targets appearing as yellow spots have equal representation of both fluorochromes and indicate no change in expression by the IR treatment. Red spots are targets increased by the treatment, and green targets are decreased. The horizontal row of green dots, landing lights, at the top and bottom are DNA labeled prior to printing on the array and serve as orientation markers for the computerized scanner. Representative target spots are identified in the enlarged segment. EST targets were prepared by PCR amplification and arrayed on poly-L-lysine coated glass slides by high speed robotic printing as previously described (DeRisi, J et al., Nat. Genet., 457-60, 1996). A complex cDNA probe was prepared from whole-cell RNA by a single round of reverse transcription in the presence of fluorescent dNTP (Cy3 dUTP or Cy5 dUTP, Amersham). Probes were hybridized to the slides for 16 hours in 3x SSC at 65oC in the presence of blockers. Hybridized slides were washed at room temperature in 0.5X SSC, 0.01% SDS, then in 0.06X SSC. The two fluorescent intensities were scanned separately using a laser confocal microscope, and the DeArray program was then used to identify target sites by image segmentation, calibrate relative ratios, and to develop confidence intervals for testing the significance of the ratios obtained (Chen, Y et al., J. Biomedical Optics, 364-374, 1997). Local background was calculated for each target location. A normalization factor was estimated from a set of 88 internal control targets (DeRisi, J et al., Nat. Genet., 457-60, 1996) with a theoretical ratio of 1.0, and the confidence interval for the array was estimated from the variance of these 88 control ratios from the expected value of 1.0. The ratios for all the targets on the array were then calibrated using the normalization factor, and ratios outside the 99% confidence interval (<0.54 or >2.37) were determined to be significantly changed by the radiation treatment. See (Amundson, SA et al., Oncogene, in press, 1999) for further explanation.