Detection of uneven hybridization using the EC series. An example of the use of external controls for detection of differential hybridization related to the location on the slide relative to the port of injection of hybridization solution on the automated station. (i) The signal intensity of the 100 μM external control is plotted as a function of sub-grid number. Sub-grids are ordered in metacolumns of 4. The higher the sub-grid number, the closer the sub-grid is to the bottom of the slide. Two arrays are plotted. In the automated hybridization station, probe is injected at the top right of each slide (ii). In Array 1, it is noted that the first point dips for every metacolumn, indicating that sub-grids on the left hand side of the slide had lower signal intensities than sub-grids on the right hand side of the slide. Similarly, the sub-grids at the bottom of the slide have lower signal intensity than sub-grids at the top of the slide. In Array 2, the pattern of differential hybridization from the top to the bottom of the slide still exists, but the positional effect is more subtle and therefore would not be detected by visualization of a slide without appropriate controls. These decreasing signal intensities may result from scanner output variables (e.g., photobleaching or optical focus as slide is scanned from top to bottom), in addition to differential hybridization.