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Multiplex in situ hybridization within a single transcript: RNAscope reveals dystrophin mRNA dynamics
- Source :
- PLoS ONE, Vol 15, Iss 9, p e0239467 (2020), PLoS ONE
- Publication Year :
- 2020
- Publisher :
- Public Library of Science (PLoS), 2020.
-
Abstract
- 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.
- Subjects :
- Male
0301 basic medicine
Muscle Physiology
Transcription, Genetic
Physiology
Muscle Proteins
Gene Expression
Biochemistry
Dystrophin
Mice
0302 clinical medicine
Transcription (biology)
Medicine and Health Sciences
Multiplex
Musculoskeletal System
In Situ Hybridization
Multidisciplinary
biology
Messenger RNA
Muscles
Muscle Biochemistry
Cell biology
Nucleic acids
medicine.anatomical_structure
Medicine
Anatomy
Research Article
Muscle tissue
Imaging Techniques
Science
DNA transcription
Muscle Tissue
In situ hybridization
Research and Analysis Methods
03 medical and health sciences
Fluorescence Imaging
Genetics
medicine
Animals
RNA, Messenger
Gene
Biology and Life Sciences
Proteins
RNA
Mice, Inbred C57BL
Cytoskeletal Proteins
Biological Tissue
030104 developmental biology
Skeletal Muscles
biology.protein
Multiplex Polymerase Chain Reaction
030217 neurology & neurosurgery
Subjects
Details
- ISSN :
- 19326203
- Volume :
- 15
- Database :
- OpenAIRE
- Journal :
- PLOS ONE
- Accession number :
- edsair.doi.dedup.....cefb9db77f51d7d14053015deb058ccd
- Full Text :
- https://doi.org/10.1371/journal.pone.0239467