Signal distributions for specific and non-specific hybridizations overlap at low absolute intensities. The median intensity of 4 B.subtilis genes (n = 24 replicates per gene × 4 = 96) was used as a linear scaling factor to balance the Cy3 and Cy5 channels. Following this normalization step, normalized intensities were Log2 transformed for efficient graphical illustration. Positive control spots (open bars) and negative control spots (filled bars) from (A) array 1 and (B) array 2 microarray hybridizations. The positive control group includes seven housekeeping genes (n = 42) and four B.subtilis genes (24 repeats per sequence; n = 96) representing sequence-specific hybridization. The negative control sequences (six repeats per sequence) include three plant genes (n = 18), three E. coli genes (n = 18), and seven human cytomegalovirus (hCMV) genes (n = 42) representing non-specific hybridization events. Data for Cy3 and Cy5 signals were pooled. Signal distributions for test genes (n = 154) from (C) array 1 and (D) array 2.