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

摘要

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 haslimited 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.