Figure 1.

Genome-wide library screen analyzed for mitotic spindle genes. (a) Outline for transfecting cells in 384-well microtiter plate format. Small interfering RNAs (siRNAs) are arrayed into 384-well microtiter plates (two siRNAs/well) and mixed with a lipid-based transfection reagent. Cell transfections are performed in a reverse or (retro)transfection manner, in which the cell culture is added to the preformed siRNA/lipid complexes and incubated at 37°C for 48 hours before -20°C methanol (MeOH) fixation. Indirect immunolocalization is used to fluorescently label cells in metaphase based on phospho-histone H3 (pHis) activity. HeLa cells were transfected in 384-well microtiter plates with 49,164 synthetic double-stranded RNAs (dsRNAs). Cells were also fluorescently labeled for α-tubulin (green), pHis (red), and DNA (blue) before being imaged on an automated microscope. (b) Plate normalized mitotic index values for the knock-down of 23,835 genes in duplicate are plotted on a Log2 scale. Calculated values are sorted in rank order (lowest to highest) and represented by the curve. Target genes with mitotic index values -2σ below the mean are shown in green. Genes with values 3σ above the mean are shown in magenta. Genes with values between the thresholds are shown in blue. Upper and lower dashed lines indicate scoring thresholds. A partial list of previously characterized genes having an essential role in chromosome segregation is given.