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FlexPCR: A streamlined multiplexed digital mRNA quantification platform with universal primers and limited
Joon Soo Park1, Jiumei Hu2, Liben Chen2
1Department of Biomedical Engineering, Johns Hopkins University, Baltimore, MD, 21218, USA.
Biosensors & Bioelectronics
|February 23, 2025
Summary
We developed Fluorescence-coding extension PCR (FlexPCR), a novel digital PCR method for accurate multiple messenger RNA (mRNA) quantification. This assay simplifies gene expression analysis and improves diagnostic capabilities.
Area of Science:
- Molecular Biology
- Genomics
- Biotechnology
Background:
- Accurate quantification of multiple messenger RNA (mRNA) targets is crucial for biomedical research and diagnostics.
- Existing PCR-based methods face limitations in multiplexing capability due to fluorescent label constraints and primer complexities, leading to amplification bias.
Purpose of the Study:
- To introduce Fluorescence-coding extension PCR (FlexPCR), a novel digital PCR assay designed for accurate and multiplexed mRNA quantification.
- To overcome the limitations of current methods by enabling detection of multiple targets using minimal fluorescence channels.
Main Methods:
- FlexPCR utilizes a universal primer and probe strategy combined with oligo extension to generate unique fluorescence-coded PCR templates for each mRNA target.
- This digital PCR-based assay simplifies assay design and reduces non-specific amplification.
Main Results:
- FlexPCR successfully quantified seven immune response mRNAs using only two fluorescence colors in human total RNA samples.
- The results demonstrated strong correlation with gold-standard single-plex RT-qPCR, validating the assay's accuracy.
Conclusions:
- FlexPCR offers a streamlined, scalable, and accurate approach for multiplexed mRNA quantification.
- This novel method has broad applications in gene expression analysis and molecular diagnostics.
