Variance, bias and background subtraction. Boxplots of the spike-in probes from the self-self experiment show increased variability with Agilent processing and with background subtraction. Each spiked-in probe is spotted 30 times on the array. All 30 replicates on each of the 4 arrays are individually plotted as well. Horizontal "jitter" was added to separate overlayed points. Bias, the distance from the reference line, is greatest for no background subtraction. The first two plots in the top row are lower intensity and this is where the increased variability with background subtraction is most apparent. Intensity increases for the remaining plots and the difference is minimal in the lower set of plots.