Effect of cDNA load and replicate qPCR on total noise levels for Rps29 (left, a and c) and Ins2 (right, b and d). Using our mathematical model, we evaluate different ways of splitting a sample before measurements. Panels (a) and (b) show how the technical noise is affected by splitting the cDNA 2-(dotted black line), 5- (dash-dotted red line) and 20-times (dashed blue line) before the qPCR. The solid green line represents the case when all cDNA generated is analyzed in one single qPCR measurement. The percentages indicate the fraction of the cDNA that is loaded to the qPCR. Panels (c) and (d) show technical noise levels as a function of initial mRNA copy number when either all cDNA (100%, solid blue line), half of the cDNA (50%, solid red line), or a third of the cDNA (33%, solid black line) is measured in one single qPCR. The dashed lines shows the combined noise levels from duplicate qPCR reactions from the cDNA split in two (red) and three (black) parts. Finally, the black dotted line shows the technical noise when the cDNA is analyzed with triplicate qPCR reactions.
Bengtsson et al. BMC Molecular Biology 2008 9:63 doi:10.1186/1471-2199-9-63