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

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dc.citation.issue3
dc.citation.volume12
dc.contributor.authorLasham A
dc.contributor.authorTsai P
dc.contributor.authorFitzgerald SJ
dc.contributor.authorMehta SY
dc.contributor.authorKnowlton NS
dc.contributor.authorBraithwaite AW
dc.contributor.authorPrint CG
dc.coverage.spatialSwitzerland
dc.date.accessioned2025-03-19T00:10:36Z
dc.date.available2025-03-19T00:10:36Z
dc.date.issued2020-03-24
dc.description.abstractTP53, 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.
dc.description.confidentialfalse
dc.edition.editionMarch 2020
dc.format.paginationE769-
dc.identifier.author-urlhttps://www.ncbi.nlm.nih.gov/pubmed/32213968
dc.identifier.citationLasham A, Tsai P, Fitzgerald SJ, Mehta SY, Knowlton NS, Braithwaite AW, Print CG. (2020). Accessing a New Dimension in TP53 Biology: Multiplex Long Amplicon Digital PCR to Specifically Detect and Quantitate Individual TP53 Transcripts.. Cancers (Basel). 12. 3. (pp. E769-).
dc.identifier.doi10.3390/cancers12030769
dc.identifier.eissn2072-6694
dc.identifier.elements-typejournal-article
dc.identifier.issn2072-6694
dc.identifier.number769
dc.identifier.piicancers12030769
dc.identifier.urihttps://mro.massey.ac.nz/handle/10179/72671
dc.languageeng
dc.publisherMDPI (Basel, Switzerland)
dc.publisher.urihttps://www.mdpi.com/2072-6694/12/3/769
dc.relation.isPartOfCancers (Basel)
dc.rights(c) 2020 The Author/s
dc.rightsCC BY 4.0
dc.rights.urihttps://creativecommons.org/licenses/by/4.0/
dc.subjectTP53 isoforms
dc.subjectalternative splicing
dc.subjectdigital PCR
dc.subjectlong RNAs
dc.subjectlong amplicon
dc.titleAccessing a New Dimension in TP53 Biology: Multiplex Long Amplicon Digital PCR to Specifically Detect and Quantitate Individual TP53 Transcripts
dc.typeJournal article
pubs.elements-id500038
pubs.organisational-groupOther
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