NEAT and tidy: a novel RT-PCR method for the independent quantification of the NEAT1_1 isoform

The long noncoding RNA NEAT1 is transcribed from a single exon gene and produces two isoforms through alternative 3'-end processing. The short polyadenylated NEAT1_1 drives proliferation in many malignancies through increasing glycolytic flux and the Warburg effect. The longer NEAT1_2 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 NEAT1_1 by subtracting NEAT1_2 from total NEAT1 levels. However, this only estimates the abundance of NEAT1_1. Standard oligo(dT)-primed RT-PCR is not suitable for quantifying NEAT1_1 as the longer NEAT1_2 sequence contains twelve poly(A) repeats, so unintended priming overestimates NEAT1_1 abundance. Here, we report the development of a novel RT-PCR method allowing relative quantification of NEAT1_1 independently of NEAT1_2. Using an anchored oligo(dT) primer for reverse transcription enriches cDNA with the NEAT1_1 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 NEAT1_1 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 in situ hybridization techniques specific to NEAT1_2-containing paraspeckles.