Figure 1: Location of controls on a 384-well plate. In a screening process, the designed biological assay is performed by using a robot to add the cell line and specific reagents (siRNA) to each well, which already contains a different oligonucleotide or control. After incubation or other required manipulations, fluorescence images are acquired and obtained for every well by an automated microscope. These raw data represent the images of each oligonucleotide or control against a specified target. (a) Generally, in a siRNA, 256 different oligonucleotides (blue) are stored in the middle of a 384-well plate, and wells on the first two and last two columns are left empty. (b) Ideally, controls should be located randomly among the 384 wells of each plate. Only the first two and the last two columns are typically available for controls. Despite this limitation, edge-related bias can be minimized by alternating the sixteen positive controls (red) and the sixteen negative controls (yellow) in the available wells, such that they appear equally on each of the sixteen rows and each of the 4 available columns.