Kinter, J. | bioRxiv Science

bioRxiv2025

ratioPCR delivers precise real-time quantification from multi-template PCR via mechanistic bias correction

Quantitative PCR (qPCR) is widely used for nucleic acid quantification but often lacks precision in detecting subtle relative changes, whereas digital PCR offers higher accuracy at reduced throughput and accessibility. Here, we introduce ratioPCR (rPCR), a bias-corrected qPCR approach that quantifies target ratios in real time by analyzing the fluorescence signal ratios during coupled amplification. Employing a mechanistic calibration model, rPCR corrects for differences in amplification efficiency, fluorophore intensity, and probe cross-reactivity, thereby achieving near-theoretical accuracy on standard qPCR platforms. Applied to alternative splicing analysis in a myotonic dystrophy type 1 model, rPCR accurately quantified isoform ratios and detected drug-induced splicing rescues in a high-throughput format. This method offers a fast, cost-effective, and scalable solution for precise relative nucleic acid quantification with potential applications in genotyping, mutation frequency estimation, and copy number variation analysis.

molecular biology↗

bioRxiv2023

Destabilization of neuromuscular junctions and deregulation of activity-dependent signalling pathways in Myotonic Dystrophy type I

Myotonic Dystrophy type I (DM1) is the most common muscular dystrophy in adults. Previous reports have highlighted that neuromuscular junctions (NMJs) deteriorate in skeletal muscle from DM1 patients and mouse models thereof. However, the underlying pathomechanisms and their contribution to muscle dysfunction remain unknown. We compared changes in NMJs and activity-dependent signalling pathways in HSALR and Mbnl1{Delta}E3/{Delta}E3 mice, two established mouse models for DM1. DM1 muscle showed major deregulation of calcium/calmodulin-dependent protein kinases II (CaMKIIs), which are key activity sensors regulating synaptic gene expression and acetylcholine receptor (AChR) recycling at the NMJ. Both mouse models displayed increased fragmentation of the endplate, which preceded muscle degeneration. Endplate fragmentation was not accompanied by changes in AChR turnover at the NMJ. However, expression of synaptic genes was up-regulated in DM1 muscle, which may be linked to the abnormally high activity of histone deacetylase 4 (HDAC4), a known target of CaMKII. Consistently, expression of myosin heavy chains was deregulated as well, leading to a major switch to type IIA fibres in Mbnl1{Delta}E3/{Delta}E3 muscle, and to a lesser extent in HSALR muscle. Interestingly, although HDAC4 was efficiently induced upon nerve injury, synaptic gene up-regulation was abrogated in DM1 muscle, together with a reduced increase in AChR turnover. This suggested that HDAC4-independent mechanisms lead to the defective response to denervation in DM1 muscle. Our study shows that activity-dependent signalling pathways are disturbed in DM1 muscle, which may contribute to NMJ destabilization and muscle dysfunction in DM1 patients.

physiology↗