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

PLoS One. 2020 Sep 24;15(9):e0239467. doi: 10.1371/journal.pone.0239467. eCollection 2020.


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 transcript molecules at sub-cellular resolution, with different target mRNAs assigned to distinct fluorophores. We instead multiplex within a single transcript, using probes targeted to the 5' and 3' regions of muscle dystrophin mRNA. Our approach shows this method can reveal transcriptional dynamics in health and disease, resolving both nascent myonuclear transcripts and exported mature mRNAs in quantitative fashion (with 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 suggest this modified multiplex ISH approach is well-suited to long genes, offering a highly tractable means to reveal complex transcriptional dynamics.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Dystrophin / genetics*
  • Dystrophin / metabolism
  • Gene Expression / genetics*
  • In Situ Hybridization / methods*
  • Male
  • Mice
  • Mice, Inbred C57BL
  • Multiplex Polymerase Chain Reaction / methods
  • Muscles / metabolism
  • RNA, Messenger / genetics
  • Transcription, Genetic / genetics


  • Dystrophin
  • RNA, Messenger

Associated data

  • figshare/10.6084/m9.figshare.9764930