title: A Study of TP53 RNA Splicing Illustrates Pitfalls of RNA-seq Methodology.
abstract: TP53 undergoes multiple RNA-splicing events, resulting in at least nine mRNA transcripts encoding at least 12 functionally different protein isoforms. Antibodies specific to p53 protein isoforms have proven difficult to develop, thus researchers must rely on the transcript information to infer isoform abundance. In this study, we used deep RNA-seq, droplet digital PCR (ddPCR), and real-time quantitative reverse transcriptase PCR (RT-qPCR) from nine human cell lines and RNA-seq data available for tumors in The Cancer Genome Atlas to analyze TP53 splice variant expression. All three methods detected expression of the FL/40TP53α_T1 variant in most human tumors and cell lines. However, other less abundant variants were only detected with PCR-based methods. Using RNA-seq simulation analysis, we determined why RNA-seq is unable to detect less abundant TP53 transcripts and discuss the implications of these findings for the general interpretation of RNA-seq data. Cancer Res; 76(24); 7151-9. ©2016 AACR.
authors.affiliation: Department of Pathology, University of Otago, Dunedin, New Zealand. sunali.mehta@otago.ac.nz.|Maurice Wilkins Centre for Molecular Biodiscovery, University of Auckland, Auckland, New Zealand.|Maurice Wilkins Centre for Molecular Biodiscovery, University of Otago, Dunedin, New Zealand.
authors.forename: Sunali
authors.initials: S
authors.lastname: Mehta
authors.affiliation: Department of Molecular Medicine and Pathology, Faculty of Medicine, University of Auckland, Auckland, New Zealand.|Bioinformatics Institute, University of Auckland, Auckland, New Zealand.
authors.forename: Peter
authors.initials: P
authors.lastname: Tsai
authors.affiliation: Maurice Wilkins Centre for Molecular Biodiscovery, University of Auckland, Auckland, New Zealand.|Maurice Wilkins Centre for Molecular Biodiscovery, University of Otago, Dunedin, New Zealand.|Department of Molecular Medicine and Pathology, Faculty of Medicine, University of Auckland, Auckland, New Zealand.
authors.forename: Annette
authors.initials: A
authors.lastname: Lasham
authors.affiliation: Children's Medical Research Institute, University of Sydney, Westmead, New South Wales, Australia.
authors.forename: Hamish
authors.initials: H
authors.lastname: Campbell
authors.affiliation: Children's Medical Research Institute, University of Sydney, Westmead, New South Wales, Australia.
authors.forename: Roger
authors.initials: R
authors.lastname: Reddel
authors.affiliation: Department of Pathology, University of Otago, Dunedin, New Zealand.|Maurice Wilkins Centre for Molecular Biodiscovery, University of Auckland, Auckland, New Zealand.|Maurice Wilkins Centre for Molecular Biodiscovery, University of Otago, Dunedin, New Zealand.|Children's Medical Research Institute, University of Sydney, Westmead, New South Wales, Australia.
authors.forename: Antony
authors.initials: A
authors.lastname: Braithwaite
authors.affiliation: Maurice Wilkins Centre for Molecular Biodiscovery, University of Auckland, Auckland, New Zealand.|Maurice Wilkins Centre for Molecular Biodiscovery, University of Otago, Dunedin, New Zealand.|Department of Molecular Medicine and Pathology, Faculty of Medicine, University of Auckland, Auckland, New Zealand.|Bioinformatics Institute, University of Auckland, Auckland, New Zealand.
authors.forename: Cristin
authors.initials: C
authors.lastname: Print
mesh_terms: D017398:Alternative Splicing / Q000235:genetics*; D045744:Cell Line, Tumor; D020869:Gene Expression Profiling / Q000379:methods; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D020033:Protein Isoforms / Q000032:analysis*; D017423:Sequence Analysis, RNA / Q000379:methods*; D016159:Tumor Suppressor Protein p53 / Q000032:analysis* / Q000235:genetics*
chemical_list: D020033:Protein Isoforms; C495901:TP53 protein, human; D016159:Tumor Suppressor Protein p53