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

Abstract

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.

Intronic TP53 Polymorphisms Are Associated with Increased Δ133TP53 Transcript, Immune Infiltration and Cancer Risk

“…RNA (1µg) extracted from the 33 cell lines was DNase I treated (Thermo Fisher Scientific, Waltham, MA, USA) and then reverse-transcribed using qScript cDNA SuperMix (Quanta Biosciences, Beverly, MA, USA), according to manufacturer’s instructions. Primers designed for specific TP53 transcript subclasses ( FL/Δ40TP53_T1 referred to as FLp53, FL/Δ40TP53_T2 referred to as Δ40p53 and Δ133TP53 , TP53α and TP53β ) were used from previous studies [ 56 , 57 ]. Absolute transcript abundance was measured with EvaGreen SuperMix using the Bio-Rad QX200 ddPCR System (Bio-Rad, Hercules, CA, USA) and converted to copies/µg RNA, as described previously [ 56 , 57 ].…”

The Δ133p53 Isoforms, Tuners of the p53 Pathway

“…Therefore, earlier clinical studies have only quantitated total expression levels of all three Δ133p53 mRNAs without distinguishing between α, β or γ transcripts ( Table 1 , upper). Recent technical developments, such as RNAscope (RNA in situ hybridization), chromatography-tandem mass spectrometry and multiplex long amplicon droplet digital PCR, are beginning to circumvent these limitations [ 36 , 37 , 38 ] ( Table 1 , lower).…”

Good Cop, Bad Cop: Defining the Roles of Δ40p53 in Cancer and Aging

“…However, this method is limited by the length of the amplicon. With the exception of one recent study [ 84 ] it has not been possible to quantitatively assess FLp53 isoform transcripts, and therefore the contribution of Δ40p53β and Δ40p53γ to cancer phenotypes and correlation with clinical outcomes remains unknown. A major limitation in detecting Δ40p53 at the protein level is the low abundance of the Δ40p53 protein relative to the FLp53 protein and the fact that there are no specific antibodies available for its analysis [ 20 ].…”