FlexPCR: A streamlined multiplexed digital mRNA quantification platform with universal primers and limited fluorescence channels

Accurate quantification of multiple messenger RNA (mRNA) targets is essential for biomedical research and disease diagnosis. Current PCR-based methods for mRNA analysis are limited by the number of fluorescent labels and the complexities associated with multiple target-specific primers, leading to amplification bias and limited multiplexing capability. Here, we introduce Fluorescence-coding extension PCR (FlexPCR), a novel digital PCR-based assay that overcomes these limitations by employing a universal primer and probe strategy in conjugation with oligo extension. This method generates unique fluorescence-coded PCR templates for each mRNA target, enabling multiplexed detection using minimal fluorescence channels. FlexPCR simplifies assay design, reduces non-specific amplification, and enhances quantification accuracy. We demonstrate the efficacy by quantifying seven immune response mRNAs using only two fluorescence colors in various human total RNA samples. The results correlate strongly with gold-standard single-plex RT-qPCR, validating the accuracy of our method. FlexPCR offers a streamlined and scalable approach for multiplexed mRNA quantification with broad applications in gene expression analysis and molecular diagnostics.