Chip-based digital PCR as a novel detection method for quantifying microRNAs in acute myocardial infarction patients

Abstract

miRNAs have shown promise as potential biomarkers for acute myocardial infarction (AMI). However, the current used quantitative real-time PCR (qRT-PCR) allows solely for relative expression of nucleic acids and it is susceptible to day-to-day variability, which has limited the validity of using the miRNAs as biomarkers. In this study we explored the technical qualities and diagnostic potential of a new technique, chip-based digital PCR, in quantifying the miRNAs in patients with AMI and ischaemia-reperfusion injury (I/R). In a dilution series of synthetic C.elegans-miR-39, chip-based digital PCR displayed a lower coefficient of variation (8.9% vs 46.3%) and a lower limit of detection (0.2 copies/μL vs 1.1 copies/μL) compared with qRT-PCR. In the serum collected from 24 patients with ST-elevation myocardial infarction (STEMI) and 20 patients with stable coronary artery disease (CAD) patients after percutaneous coronary intervention (PCI), we used qRT-PCR and multiplexed chip-based digital PCR to quantify the serum levels of miRNA-21 and miRNA-499 as they have been validated in AMI in prior studies. In STEMI, I/R injury was assessed via measurement of ST-segment resolution (ST-R). Chip-based digital PCR revealed a statistical significance in the difference of miR-21 levels between stable CAD and STEMI groups (118.8 copies/μL vs 59 copies/μL; P=0.0300), whereas qRT-PCR was unable to reach significance (136.4 copies/μL vs 122.8 copies/μL; P=0.2273). For miR-499 levels, both chip-based digital PCR and qRT-PCR revealed statistically significant differences between stable CAD and STEMI groups (2 copies/μL vs 8.5 copies/μL, P=0.0011; 0 copies/μL vs 19.4 copies/μL; P<0.0001). There was no association between miR-21/499 levels and ST-R post-PCI. Our results show that the chip-based digital PCR exhibits superior technical qualities and promises to be a superior method for quantifying miRNA levels in the circulation, which may become a more accurate and reproducible method for directly quantifying miRNAs, particularly for use in large multi-centre clinical trials.

Keywords: ST-segment elevation myocardial infarction; chip-based digital PCR; ichaemia-reperfusion injury; micro-RNAs; qRT-PCR.

Figure 1

Technical qualities of chip-based digital PCR compared to qRT-PCR, including coefficient of variation (CV%), linearity, limit of detection (LOD), limit of quantification (LOQ) and lower limit of linear range (LLLR) for a dilution series of C.elegans-miR-39 performed in duplicate. (A) Trendline comparing coefficient of variation (CV%) for qRT-PCR and chip-based digital PCR at different concentration points with corresponding table demonstrating average CV% and difference in CV% (B) Duplicate measurements are presented log transformed as absolute copies for each dilution point reported for qRT-PCR and chip-based digital PCR with corresponding linearity and goodness-of-fit (r ²-value). Data are presented as mean±SD (C) LOD, LOQ and LLLR for qRT-PCR and chip-based digital PCR.

Figure 2

Quantification of circulating miRNA-21-5p (A). Duplicate measurements of miR-21 for each patient sample represented as median and IQR between stable CAD and STEMI groups with corresponding P-value for qRT-PCR (blue) and chip-based digital PCR (red) as calculated by a Mann-Whitney U test. (B) ROC analysis from data in A, representing the senstivity and specificity for cTnT, qRT-PCR and chip-based digital PCR with corresponding area under the curve values. (C) Median and IQR of all measurements by qRT-PCR and chip-based digital PCR for miRNA-21 for STEMI patients and patients with stable CAD given in copies/μL.

Figure 3

Quantification of circulating miRNA-499-5p (A). Duplicate measurements of miR-499 for each patient sample represented as median and IQR between stable CAD and STEMI groups with corresponding P-value for qRT-PCR (blue) and chip-based digital PCR (red) as calculated by a Mann-Whitney U test. (B) ROC analysis from data in A, representing the senstivity and specificity for cTnT, qRT-PCR and chip-based digital PCR with corresponding area under the curve values. (C) Median and IQR of all measurements by qRT-PCR and chip-based digital PCR for miR-499 for STEMI patients and patients with stable CAD given in copies/μL.

Figure 4

Chip-based digital PCR results of miRNA-21 and 499 levels in diagnosing ischaemia-reperfusion injury as defined by ST-segment resolution (ST-R) <70%. (A) Median and IQR of miR-21 (black) and miR-499 (blue) levels with corresponding significance values calculated via a Mann-Whitney U test. (B) ROC analysis of miR-21 and miR-499 levels in diagnosing I/R injury.