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Development and Evaluation of a Droplet Digital PCR Assay for the Accurately Detecting the CircHIPK3 in Plasma
Yuanye Ji1, Ping Tuo1, Shun Zhang1
1Department of Medical Laboratory, Ningbo No.2 Hospital, Ningbo City, Zhejiang Province, P.R. China.
Insights
Droplet digital PCR (ddPCR) offers a highly sensitive method for detecting circular RNA HIPK3 (circHIPK3) in plasma. This advanced technique improves upon quantitative PCR (qPCR) for early hepatocellular carcinoma (HCC) detection.
Area of Science:
- Oncology
- Molecular Biology
- Biomarker Discovery
Background:
- Hepatocellular carcinoma (HCC) is a growing global health concern.
- Circular RNA HIPK3 (circHIPK3) is implicated in HCC development and serves as a potential biomarker.
Purpose of the Study:
- To develop a sensitive and accurate droplet digital PCR (ddPCR) method for circHIPK3 detection in low-load plasma samples.
- To compare the performance of ddPCR with quantitative PCR (qPCR) for circHIPK3 quantification.
Main Methods:
- Design and optimization of circHIPK3 gradient primers and probes for ddPCR.
- Assessment of linearity and sensitivity using circHIPK3 plasmid DNA.
- Quantitative determination of circHIPK3 in cell lines and human plasma samples.
Main Results:
- ddPCR demonstrated superior linearity (R²=0.9940) compared to qPCR (R²=0.9877).
- ddPCR exhibited significantly lower limits of detection (LOD=0.594 copies/μl) than qPCR (LOD=5.753 copies/μl).
- ddPCR identified 11 additional positive samples missed by qPCR, showing strong correlation (Kappa=0.677, ICC=0.903).
Conclusions:
- The ddPCR-based method for circHIPK3 detection is highly sensitive and accurate.
- This ddPCR approach shows significant potential for clinical applications in HCC liquid biopsy.
Abstract:
Hepatocellular carcinoma (HCC) is an increasingly prevalent malignant neoplasm on a global scale. Circrna HIPK3 (circHIPK3) has been identified as playing a key role in HCC tumorigenesis and as a novel biomarker. In this study, we aimed at developing a sensitive and accurate method for the detection of circHIPK3 in low load plasma samples using a droplet digital PCR (ddPCR). We designed circHIPK3 gradient primers and probes and optimized the PCR system to improve performance. Then we assessed and compared the linearity and sensitivity of ddPCR and quantitative PCR (qPCR) using the circHIPK3 plasmid DNA as a template. Using these methods, circHIPK3 concentrations were quantitatively determined in 3 cell lines and 40 plasma samples to assess clinical stability. Within the plasmid concentration range of 31-36 copies/μl, the ddPCR exhibited a linear fitting equation of Y = 1.037X-0.1724 with R² value of 0.9940, which surpassed the corresponding R² value of 0.9877 for qPCR. Furthermore, the limit of blank (LOB) and limit of detection (LOD) for ddPCR were determined to be 0.157 copies/μl and 0.594 copies/μl, respectively, which were significantly lower than the LOD of qPCR (5.753 copies/μl). In clinical samples, ddPCR demonstrated a commendable correlation with qPCR, evidenced by a Kappa value of 0.677 (p < 0.05, 95% CI [0.503-0.851]) and an intraclass correlation coefficient (ICC) of 0.903 (95% CI [0.831-0.946]). Notably, ddPCR identified 11 positive samples that qPCR failed to detect. The ddPCR-based circHIPK3 liquid biopsy method emerged as a highly sensitive and accurate approach, lending itself well to clinical applications.
