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Updated: Jan 12, 2026
MicroRNA Based Liquid Biopsy: The Experience of the Plasma miRNA Signature Classifier MSC for Lung Cancer Screening
Published on: October 26, 2017
A robust ddPCR assay for the absolute quantification of miR-192-5p in hepatocellular carcinoma liquid biopsies
Xinmiao Liu1, Yefan Zhang2, Narisu Narisu3
1Department of Clinical Laboratory, State Key Laboratory of Molecular Oncology, National Cancer Center/National Clinical Research Center for Cancer/Cancer Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China.
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.
