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

Samuel ROBINSON1,2, Marie FOLLO3, David HAENEL1, Maximilian MAULER4, Daniela STALLMANN1, Lukas Andreas HEGER1, Thomas HELBING1, Daniel DUERSCHMIED1, Karlheinz PETER2,5, Christoph BODE1, Ingo AHRENS1,6, Marcus HORTMANN1
1 Department of Cardiology and Angiology I, Heart Center Freiburg University, Faculty of Medicine, University of Freiburg, Germany
2 Department of Medicine, Monash University, Melbourne, Australia
3 Department of Medicine I, Lighthouse Core Facility, Medical Center - University of Freiburg, Faculty of Medicine, University of Freiburg, Freiburg, Germany
4 Faculty of Biology, University of Freiburg
5 Baker IDI Heart and Diabetes Institute, Melbourne, Australia
6 Augustinerinnen Hospital, Academic Teaching Hospital University of Cologne, Cologne, Germany
Correspondence to: Marcus HORTMANN:,
DOI: 10.1038/aps.2017.136
Received: 21 July 2017
Accepted: 10 October 2017
Advance online: 30 November 2017


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; ichaemia-reperfusion injury; micro-RNAs; chip-based digital PCR; qRT-PCR

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