S Robinson1, M Follo2, D Haenel3, M Mauler3, D Stallmann3, M Tewari4, D Duerschmied3, K Peter5, C Bode3, I Ahrens6, M Hortmann7. 1. Department of Cardiology and Angiology I, Heart Center Freiburg University, Faculty of Medicine, University of Freiburg, Germany; Department of Medicine, Monash University, Melbourne, Australia. 2. Department of Medicine I, Lighthouse Core Facility, Medical Center - University of Freiburg, Faculty of Medicine, University of Freiburg, Germany. 3. Department of Cardiology and Angiology I, Heart Center Freiburg University, Faculty of Medicine, University of Freiburg, Germany. 4. Department of Internal Medicine, Hematology/Oncology Division, University of Michigan Medical School, Ann Arbor, MI, USA; Department of Biomedical Engineering, University of Michigan, Ann Arbor, MI, USA; Biointerfaces Institute, University of Michigan, Ann Arbor, MI, USA; Center for Computational Medicine and Bioinformatics, University of Michigan, Ann Arbor, MI, USA; Baker IDI Heart and Diabetes Institute, Melbourne, Australia. 5. Department of Medicine, Monash University, Melbourne, Australia; Baker IDI Heart and Diabetes Institute, Melbourne, Australia. 6. Department of Cardiology and Angiology I, Heart Center Freiburg University, Faculty of Medicine, University of Freiburg, Germany; Department of Cardiology, Augustinerinnen Hospital, Academic Teaching Hospital, University of Cologne, 50678 Cologne, Germany. 7. Department of Cardiology and Angiology I, Heart Center Freiburg University, Faculty of Medicine, University of Freiburg, Germany. Electronic address: marcus.hortmann@universitaets-herzzentrum.de.
Abstract
BACKGROUND: micro-RNAs have shown promise as potential biomarkers for acute myocardial infarction and ischemia-reperfusion injury (I/R). Most recently droplet digital polymerase chain reaction (ddPCR) has been introduced as a more reliable and reproducible method for detecting micro-RNAs. AIMS: We aimed to demonstrate the improved technical performance and diagnostic potential of ddPCR by measuring micro-RNAs in ST-elevation myocardial infarction (STEMI). METHODS: A dilution series was performed in duplicate on synthetic Caenorrhabditis elegans-miR-39, comparing quantitative real-time PCR (qRT-PCR) and ddPCR. We used ddPCR and qRT-PCR to quantify the serum levels of miR-21, miR-208a and miR-499 between STEMI patients (n=24) and stable coronary artery disease (CAD) patients (n=20). In STEMI, I/R injury was assessed via measurement of ST-segment resolution. RESULTS: In the dilution series, ddPCR demonstrated superior coefficient of variation (12.1%vs.32.9%) and limit of detection (0.9325 vs.2.425copies/μl). In the patient cohort, ddPCR demonstrated greater differences in miR-21 levels (2190.5 vs. 484.7copies/μl; p=0.0004 for ddPCR and 136.4 vs. 122.8copies/μl; p=0.2273 for qRT-PCR) and in miR-208a (0 vs. 24.1copies/μl, p=0.0013 for ddPCR and 0 vs. 0copies/μl, p=0.0032 for qRT-PCR), with similar differences observed in miR-499 levels (9.4 vs. 81.5copies/μl, p<0.0001 for ddPCR and 0 vs. 19.41copies/μl, p<0.0001 for qRT-PCR). ddPCR also more accurately defined STEMI for all miRNAs (area under the curve (AUC) of 0.8021/0.7740/0.9063 for miR-21/208a/499 with ddPCR vs. AUC of 0.6083/0.6917/0.8417 with qRT-PCR). However, there was no association between miR-21/208a/499 levels and ischemia-reperfusion injury. CONCLUSION: ddPCR demonstrates superiority in both technical performance and diagnostic potential compared to qRT-PCR. Ultimately, this supports its use as a diagnostic method for quantifying micro-RNAs, particularly in large multi-center trials.
BACKGROUND: micro-RNAs have shown promise as potential biomarkers for acute myocardial infarction and ischemia-reperfusion injury (I/R). Most recently droplet digital polymerase chain reaction (ddPCR) has been introduced as a more reliable and reproducible method for detecting micro-RNAs. AIMS: We aimed to demonstrate the improved technical performance and diagnostic potential of ddPCR by measuring micro-RNAs in ST-elevation myocardial infarction (STEMI). METHODS: A dilution series was performed in duplicate on synthetic Caenorrhabditis elegans-miR-39, comparing quantitative real-time PCR (qRT-PCR) and ddPCR. We used ddPCR and qRT-PCR to quantify the serum levels of miR-21, miR-208a and miR-499 between STEMI patients (n=24) and stable coronary artery disease (CAD) patients (n=20). In STEMI, I/R injury was assessed via measurement of ST-segment resolution. RESULTS: In the dilution series, ddPCR demonstrated superior coefficient of variation (12.1%vs.32.9%) and limit of detection (0.9325 vs.2.425copies/μl). In the patient cohort, ddPCR demonstrated greater differences in miR-21 levels (2190.5 vs. 484.7copies/μl; p=0.0004 for ddPCR and 136.4 vs. 122.8copies/μl; p=0.2273 for qRT-PCR) and in miR-208a (0 vs. 24.1copies/μl, p=0.0013 for ddPCR and 0 vs. 0copies/μl, p=0.0032 for qRT-PCR), with similar differences observed in miR-499 levels (9.4 vs. 81.5copies/μl, p<0.0001 for ddPCR and 0 vs. 19.41copies/μl, p<0.0001 for qRT-PCR). ddPCR also more accurately defined STEMI for all miRNAs (area under the curve (AUC) of 0.8021/0.7740/0.9063 for miR-21/208a/499 with ddPCR vs. AUC of 0.6083/0.6917/0.8417 with qRT-PCR). However, there was no association between miR-21/208a/499 levels and ischemia-reperfusion injury. CONCLUSION: ddPCR demonstrates superiority in both technical performance and diagnostic potential compared to qRT-PCR. Ultimately, this supports its use as a diagnostic method for quantifying micro-RNAs, particularly in large multi-center trials.
Authors: Louise Benning; Samuel Robinson; Marie Follo; Lukas Andreas Heger; Daniela Stallmann; Daniel Duerschmied; Christoph Bode; Ingo Ahrens; Marcus Hortmann Journal: J Vis Exp Date: 2018-07-03 Impact factor: 1.355
Authors: Linsey J F Peters; Jürgen Floege; Erik A L Biessen; Joachim Jankowski; Emiel P C van der Vorst Journal: Int J Mol Sci Date: 2020-09-07 Impact factor: 5.923