| Autor: |
Lasham A; Department of Molecular Medicine and Pathology, School of Medical Sciences, University of Auckland, Auckland 1142, New Zealand.; Maurice Wilkins Centre, University of Auckland, Auckland 1010, New Zealand., Tsai P; Department of Molecular Medicine and Pathology, School of Medical Sciences, University of Auckland, Auckland 1142, New Zealand.; Maurice Wilkins Centre, University of Auckland, Auckland 1010, New Zealand., Fitzgerald SJ; Department of Molecular Medicine and Pathology, School of Medical Sciences, University of Auckland, Auckland 1142, New Zealand.; Maurice Wilkins Centre, University of Auckland, Auckland 1010, New Zealand., Mehta SY; Maurice Wilkins Centre, University of Auckland, Auckland 1010, New Zealand.; Department of Pathology, University of Otago, Dunedin 9016, New Zealand., Knowlton NS; Department of Molecular Medicine and Pathology, School of Medical Sciences, University of Auckland, Auckland 1142, New Zealand.; Maurice Wilkins Centre, University of Auckland, Auckland 1010, New Zealand., Braithwaite AW; Maurice Wilkins Centre, University of Auckland, Auckland 1010, New Zealand.; Department of Pathology, University of Otago, Dunedin 9016, New Zealand.; Malaghan Institute of Medical Research, Wellington 6242, New Zealand., Print CG; Department of Molecular Medicine and Pathology, School of Medical Sciences, University of Auckland, Auckland 1142, New Zealand.; Maurice Wilkins Centre, University of Auckland, Auckland 1010, New Zealand. |
| Abstrakt: |
TP53 , the most commonly-mutated gene in cancer, undergoes complex alternative splicing. Different TP53 transcripts play different biological roles, both in normal function and in the progression of diseases such as cancer. The study of TP53's alternative RNA splice forms and their use as clinical biomarkers has been hampered by limited specificity and quantitative accuracy of current methods. TP53 RNA splice variants differ at both 5' and 3' ends, but because they have a common central region of 618 bp, the individual TP53 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 TP53 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 TP53 transcripts are co-expressed in breast cancers, and illustrate the potential for this method to identify novel TP53 transcripts in tumour cells. This capability will facilitate a new level of biological and clinical understanding of the alternatively-spliced TP53 isoforms. |
