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.