Digital Droplet PCR for the Absolute Quantification of Exon Skipping Induced by Antisense Oligonucleotides in (Pre-)Clinical Development for Duchenne Muscular Dystrophy
Fig 3
Temperature gradient ddPCR for both DMD ex51-52.2 and DMD ex47-52 assays.
(A) DMD ex51-52.2 assay and (B) DMD ex47-52 assay. The left panels show how the positive droplet populations change with decreasing annealing temperatures. At higher annealing temperatures the overall fluorescence of the positive droplet population (in blue) is lower, caused by a less efficient PCR amplification, and consequently the separation of the positive and negative droplet (in grey) clouds is less pronounced. In the right panels the corresponding concentrations of the constructs along the temperature gradient are represented. The %CV (coefficient of variation) between the different annealing temperatures is 3–4%, indicating a highly consistent quantification of the construct concentration, even at suboptimal amplification conditions at then higher annealing temperatures. Data is represented as mean with 95% CI (right panel).