The Pitfalls of RNA-Seq Methodology

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, researchers from the University of Otago 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, the researchers 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.

Accurate quantitation of transcripts by RSEM is dependent on
RNA-seq read depth and relative abundance

rna-seq

Mehta S, Tsai P, Lasham A, Campbell H, Reddel R, Braithwaite A, Print C. (2016) A Study of TP53 RNA Splicing Illustrates Pitfalls of RNA-seq Methodology. Cancer Res [Epub ahead of print]. [abstract]

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