Digital Droplet PCR for the Absolute Quantification of Exon Skipping Induced by Antisense Oligonucleotides in (Pre-)Clinical Development for Duchenne Muscular Dystrophy
Fig 2
Design and specificity of the Taqman assays representative for the exon 48–50 deletion transcripts in ddPCR analysis.
(A) Position of Taqman MGB assays that were designed to detect exon 51 skipping in a muscle cell culture from a DMD patient with a deletion of exons 48–50 (Δ48–50). Primers and exon spanning probes were positioned as indicated by the arrows and the red lines. (B) The specificity of both DMD ex51-52.2 and DMD ex47-52 Taqman assays was confirmed by ddPCR analysis using dystrophin cDNA constructs representative for Δ48–50 transcript fragments with or without exon 51. The left panel shows the droplet populations obtained using both assays. The ex51-52.2 assay generated positive droplets when the construct containing exon 51 was used as template, but not when the exon 51 lacking construct was the template. For the ex47-52 assay detection was vice versa, yielding positive droplets using the exon 51 lacking template. The red arrow (in left panel) indicates a cloud of droplets with low fluorescence, arising from partial binding of the Taqman probe used in this assay to exon 47 and/or 52 sequences present but not joined in the template containing exon 51. The right panel shows the concentration of the constructs that was calculated based on the number of positive droplets for both assays. Data is represented as mean with 95% confidence interval (CI) (right panel).