Written by Edward Mabry
Week 21 (7/2/21) Dystrophin is an incredibly long gene of critical importance to crippling diseases such as Duchenne muscular dystrophy, but due to the limitations of traditional in situ hybridization (ISH) methods, the mRNA dynamics remained difficult to study. We recently discussed a research article from the Comparative Neuromuscular Diseases Laboratory validating RNAscope as a method to reveal the complex transcriptional dynamics of dystrophin mRNA. Hildyard et al. utilizes this sensitive ISH method for quantifying the dp247 isoform in mouse skeletal muscle localized both in the nucleus and sarcoplasm, possible in part to RNAscope’s reduction to off-targeting and its use of amplifiers for depositing high concentrations of fluorophore at the probe site. Probes were developed for both the 5’ and 3’ ends of the dystrophin mRNA such that detection and differentiation of the nascent and mature dp247 was possible at a subcellular level for individual transcripts. Quantification of the transcripts by RNAscope, in addition with support by qPCR analysis, implied that even in healthy mice the mature dp247 transcripts had a half-life of about 2 to 3 hours, even though the transcription of one such mRNA took about 16 hours. The researchers believe that this is due to the supply of transcripts for muscular dystrophin being higher than demand, but in dystrophic mice the transcription initiation events are less frequent, leading to fewer 5’ nascent signals and then prompt degradation following nuclear export due to premature termination codon and the NMD pathway. Overall, this ISH method shows promise towards quantifying long transcripts with low mRNA expression, such that complex transcriptional pathways could be more readily perceived and understood.