Article

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

Authors: 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: marcus.hortmann@universitaets-herzzentrum.de,
DOI: 10.1038/aps.2017.136
Received: 21 July 2017
Accepted: 10 October 2017
Advance online: 30 November 2017

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

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