A robust ddPCR assay for the absolute quantification of miR-192-5p in hepatocellular carcinoma liquid biopsies
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Xinmiao Liu
and
Hongjun Gao
Abstract
Objectives
MicroRNA-192-5p, a liver-enriched miRNA downregulated in hepatocellular carcinoma (HCC), is a promising biomarker, but its clinical use is limited by technical challenges in detecting low-abundance plasma miRNAs. This study innovatively uses droplet digital PCR (ddPCR) with locked nucleic acid (LNA)-modified probes to develop an ultrasensitive and standardized method for miRNA quantification in liquid biopsies.
Methods
Following Minimum Information for Publication of Quantitative Real-Time PCR Experiments guidelines, seven primer-probe combinations were screened by qPCR, and one with the lowest Ct variability (Ct <35) was selected. LNA-modified Probe P-2 was designed to enhance target hybridization. Reaction conditions were optimized to 1 μM primers, 300 nM probes, and 55 cycles. Analytical validation included trueness, precision, sensitivity, linear range, and interference testing. Plasma from 87 HCC patients and 57 controls was analyzed, and a logistic model combining miR-192-5p, AFP, and AFU was evaluated.
Results
The LNA probe improved positive droplet counts by 32 %. The dPCR showed excellent precision (intra-batch CV 2.31–21.63 %, inter-batch 17.54 %) and trueness (R=0.92 vs. RT-qPCR). Sensitivity thresholds were LoB=1.75, LoD=3.33, LoQ=13.45 copies/μL, with linear range 13.45–129,693 copies/μL (R2=0.9965). It tolerated low hemoglobin and triglycerides but was affected by bilirubin. HCC patients had lower miR-192-5p (444.2 vs. 753.5 copies/μL, p<0.001), with AUC=0.70. The multi-marker model had AUC=0.88.
Conclusions
This LNA-optimized ddPCR assay resolves miRNA liquid biopsy barriers. The combinatorial model outperforms single biomarkers, offering a clinical tool for the precise quantification of HCC-specific miRNAs. Standardized workflows ensure reproducibility, and multicenter studies are needed for validation.
Funding source: the Science and Technology Department Basic Research Project of Shanxi
Award Identifier / Grant number: 202303021211225
Funding source: Scientific Research Support Program for High-Level Talent and Team Recruitment in Public Institutions from Department of Human Resources and Social Security of Inner Mongolia Autonomous Region
Award Identifier / Grant number: 2024-124
Funding source: CAMS Innovation Fund for Medical Sciences
Award Identifier / Grant number: 2022-I2M-C&T-B-080
Acknowledgments
We thank the colleagues in Department of Clinical Laboratory and State Key Laboratory of Molecular Oncology, Cancer Hospital, Chinese Academy of Medical Sciences, and in Department of Clinical Laboratory of Shanxi Province Cancer Hospital.
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Research ethics: All procedures were in accordance with the Helsinki Declaration. The protocol was reviewed and approved by the Ethics Committee of the National Cancer Center/Cancer Hospital, Chinese Academy of Medical Sciences (Approval No.: NCC2022C-524). All samples used were exclusively anonymized samples.
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Informed consent: Not applicable.
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Author contributions: Xinmiao Liu: Writing–original draft, Visualization, Methodology, Formal analysis. Yefan Zhang: Formal analysis, Data curation, Writing–original draft. Narisu: Formal analysis, Data curation, Methodology, Writing–original draft. Fan Wu: Data curation, Methodology, Writing–original draft. Ke Zhang: Visualization, Writing–original draft. Yulin Sun: Conceptualization, Project administration, Investigation, Supervision, Writing–original draft. Hongjun Gao: Conceptualization, Formal analysis, Funding acquisition, Investigation, Resources, Supervision, Writing–original draft, Writing–review and editing. All authors have accepted responsibility for the entire content of this manuscript and approved its submission.
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Use of Large Language Models, AI and Machine Learning Tools: None declared.
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Conflict of interest: The authors state no conflict of interest.
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Research funding: The authors declare financial support was received for the research, authorship, and/or publication of this article. This work was supported by grants from the CAMS Innovation Fund for Medical Sciences (2022-I2M-C&T-B-080), the Science and Technology Department Basic Research Project of Shanxi (202303021211225) and Scientific Research Support Program for High-Level Talent and Team Recruitment in Public Institutions from Department of Human Resources and Social Security of Inner Mongolia Autonomous Region(2024-124).
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Data availability: The raw data can be obtained on request from the corresponding author.
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Supplementary Material
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