Multiplex in situ hybridization within a single transcript: RNAscope reveals dystrophin mRNA dynamics

Dystrophin plays a vital role in maintaining muscle health, yet low mRNA expression, lengthy transcription time and the limitations of traditional in-situ hybridization (ISH) methodologies mean that the dynamics of dystrophin transcription remain poorly understood. RNAscope is highly sensitive ISH method that can be multiplexed, allowing detection of individual transcripts at sub-cellular resolution, with different target mRNAs assigned to distinct fluorophores. We present a novel approach, instead using RNAscope probes targeted to 5' and 3' regions of the same transcript: labelling muscle dystrophin mRNA in this manner allows transcriptional dynamics to be deciphered in health and disease, resolving both nascent myonuclear transcripts and exported mature mRNAs (the latter absent in dystrophic muscle, yet restored following therapeutic intervention). We show that even in healthy muscle, immature dystrophin mRNA predominates (60-80% of total), with the surprising implication that the half-life of a mature transcript is markedly shorter than the time invested in transcription: at the transcript level, supply may exceed demand. Our findings provide unique spatiotemporal insight into the behaviour of this long transcript (with implications for therapeutic approaches), and further suggests this modified multiplex ISH approach is well-suited to long genes, offering a highly tractable means to reveal complex transcriptional dynamics.