Figure 1

Visualisation of the different steps in a typical digital PCR workflow. Important variance components are included as arrows between the appropriate steps. The steps are: (1) extracting RNA or DNA from the biological sample, (2) preparing the PCR master mix and including a quantity of extract, (3) dividing the reaction mix over a large number of partitions (droplets or cells), (4) amplifying the target material present in the partitions over a selected number of amplification cycles and measuring the endpoint fluorescence and (5) estimating the target concentration and quantifying the uncertainty on the estimates. Variance components are (i) technical variation: sampling variation and pipette error, (ii) machine-specific variation: unequal partition size and possible partition loss, and (iii) possibly user-optimized (mis)classification of endpoint fluorescence.