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and tidy: a novel RT-PCR method for the independent quantification of the isoform.

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Biology methods & protocols 2025 Vol.10(1) p. bpaf073
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Smith NE, Petersen J, Michael MZ

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The long noncoding RNA is transcribed from a single exon gene and produces two isoforms through alternative 3'-end processing.

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APA Smith NE, Petersen J, Michael MZ (2025). and tidy: a novel RT-PCR method for the independent quantification of the isoform.. Biology methods & protocols, 10(1), bpaf073. https://doi.org/10.1093/biomethods/bpaf073
MLA Smith NE, et al.. " and tidy: a novel RT-PCR method for the independent quantification of the isoform.." Biology methods & protocols, vol. 10, no. 1, 2025, pp. bpaf073.
PMID 41230023 ↗

Abstract

The long noncoding RNA is transcribed from a single exon gene and produces two isoforms through alternative 3'-end processing. The short polyadenylated drives proliferation in many malignancies through increasing glycolytic flux and the Warburg effect. The longer lacks a poly(A)-tail but is an essential scaffold for nuclear paraspeckles, nuclear condensates that reportedly play a tumour protective role. Due to the two isoforms sharing identical 5'-ends, many previous studies have quantified by subtracting from total levels. However, this only estimates the abundance of Standard oligo(dT)-primed RT-PCR is not suitable for quantifying as the longer sequence contains twelve poly(A) repeats, so unintended priming overestimates abundance. Here, we report the development of a novel RT-PCR method allowing relative quantification of independently of Using an anchored oligo(dT) primer for reverse transcription enriches cDNA with the isoform, and the use of a longer primer anchoring sequence at the PCR stage enhances detection specificity. Our method is validated by the successful independent quantification of following a forced isoform switch using antisense oligomers in both cancer and non-cancer cell lines. Additionally, we have visualized this isoform switch in colorectal cancer cell lines using fluorescent hybridization techniques specific to -containing paraspeckles.

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