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.

RETRACTED ARTICLE: Overexpression of microRNA-202-3p protects against myocardial ischemia-reperfusion injury through activation of TGF-β1/Smads signaling pathway by targeting TRPM6

“…Myocardial I/R injury is a pathological process that results in DNA, plasma membrane and and protein damage, contributing to severe myocardial injury and inflicting pain and damage on I/R injury victims [32]. Robinson et al have revealed that miRNAs are being studied more in depth due to their uses as potential markers for acute myocardial infarction (AMI), as some specific miRNAs may be associated with AMI and I/R injury [33]. Investigations on functional roles of miRNAs in I/R injury and ventricular remodeling may allow us to understand more as to how miRNAs affect cellular behaviors, such as cell apoptosis, fibrosis or infarction.…”

Potential Clinical Implications of miR-1 and miR-21 in Heart Disease and Cardioprotection

“…Importantly, detected levels of (not only) miR-21 may depend on the method of miR measurement. For instance, miR-1 levels statistically differed between STEMI patients and patients with stable coronary artery disease when quantified using novel chip-based digital PCR whereas classical qRT-PCR was unable to reach significance [79]. Another novel method, droplet digital PCR, detected no association between miR-21 levels and ischemia-reperfusion injury in STEMI patients [80], and also did not find miR-21 as a potentially relevant marker of coronary artery disease [81].…”

Circulating biomarkers for cardiovascular diseases: the beats never stop

“…Accordingly, this Special Issue of Acta Pharmacologica Sinica was planned to provide a forum for high quality original research and review articles to showcase the most recent advances in translational research and clinical practices on a wide-spectrum array of conventional as well as novel cardio-vascular biomarkers. Coverage of this Special Issue encompasses various forms of large or small molecules, such as proteins, peptides, cytokines, microRNAs, and metabolic by- This Special Issue covers a variety of pathological types of CVD, including acute myocardial infarction (AMI) [2][3][4][5] , heart failure [6] , aortic aneurysms [7,8] , carotid restenosis [9] , pulmonary arterial hypertension (PAH) [10] , and cardiac amyloidosis [11] . Several review articles provided in-depth analyses on the important diagnostic and prognostic roles of zinc [12] , oxylipins [13] , and leptin [14] in the fundamental cellular signaling and pathogenic processes in CVD and diabetes.…”

“…Several review articles provided in-depth analyses on the important diagnostic and prognostic roles of zinc [12] , oxylipins [13] , and leptin [14] in the fundamental cellular signaling and pathogenic processes in CVD and diabetes. Notably, all the 7 original research articles are clinical research on Asian patients with AMI [5] or PAH [10] as well as European patients with AMI [2] , chronic heart failure [6] , vascular aneurysm [7] or restenosis [9] , and stroke [15] .…”

“…It is noteworthy that 4 review articles [4,[16][17][18] and 2 original research papers [2,10] in this Special Issue are focusing on a cutting-edged topic concerning the potential roles of circulating noncoding RNAs (ncRNAs), which include microRNAs (miRNAs), long noncoding RNAs (lncRNAs) and circular RNAs (circRNAs), in serving as a new generation of biomarkers for CVD. Among these elegant works, Bayoumi et al reviewed the circular noncoding RNAs as potential therapies and circulating biomarkers for CVD [18] .…”