Accessing a New Dimension in TP53 Biology: Multiplex Long Amplicon Digital PCR to Specifically Detect and Quantitate Individual Transcripts.

, the most commonly-mutated gene in cancer, undergoes complex alternative splicing. Different transcripts play different biological roles, both in normal function and in the progression of diseases such as cancer. The study of alternative RNA splice forms and their use as clinical biomarkers has been hampered by limited specificity and quantitative accuracy of current methods. RNA splice variants differ at both 5' and 3' ends, but because they have a common central region of 618 bp, the individual transcripts are impossible to specifically detect and precisely quantitate using standard PCR-based methods or short-read RNA sequencing. Therefore, we devised multiplex probe-based long amplicon droplet digital PCR (ddPCR) assays, which for the first time allow precise end-to-end quantitation of the seven major transcripts, with amplicons ranging from 0.85 to 1.85 kb. Multiple modifications to standard ddPCR assay procedures were required to enable specific co-amplification of these long transcripts and to overcome issues with secondary structure. Using these assays, we show that several transcripts are co-expressed in breast cancers, and illustrate the potential for this method to identify novel transcripts in tumour cells. This capability will facilitate a new level of biological and clinical understanding of the alternatively-spliced isoforms.