Design considerations and validation strategies for droplet digital PCR assays targeting exon junctions and rare splice-variant transcripts

Droplet digital PCR is widely recognised for its sensitivity, reproducibility and precision in quantifying target nucleic acid fragments. This technology is crucial in validating gene expression changes across various experimental settings. In particular, droplet digital PCR is highly effective for monitoring the molecular consequences of splice-modulating antisense oligonucleotides when assays are appropriately designed. Using a hydrolysis probe targeting specific exon–exon junctions and EvaGreen assays, we present primer and probe designs, along with troubleshooting strategies, to accurately detect transcripts missing exons and rare transcript variants. We also discuss key experimental considerations when transitioning from in vitro cell culture studies to in vivo molecular analyses for quantifying exon junctions. Additionally, we highlight the challenges in designing probes to detect exon junctions that are highly similar to other junctions.