Droplet digital PCR as a novel detection method for quantifying microRNAs in acute myocardial infarction

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/mu l). In the patient cohort, ddPCR demonstrated greater differences inmiR-21 levels (2190.5 vs. 484.7 copies/mu l; p = 0.0004 for ddPCR and 136.4 vs. 122.8 copies/mu l; p = 0.2273 for qRT-PCR) and in miR-208a (0 vs. 24.1 copies/mu l, p = 0.0013 for ddPCR and 0 vs. 0 copies/mu l, p = 0.0032 for qRT-PCR), with similar differences observed in miR-499 levels (9.4 vs. 81.5 copies/mu l, p < 0.0001 for ddPCR and 0 vs. 19.41 copies/mu l, 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 qRTPCR). 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 diagnosticmethod for quantifyingmicro-RNAs, particularly in large multi-center trials. (C) 2017 Elsevier B.V. All rights reserved

Influence of extracorporeal membrane oxygenation on serum microRNA expression

Objective To date, no biomarkers have been established to predict haematological complications and outcomes of extracorporeal membrane oxygenation (ECMO). The aim of this study was to investigate the expression of a panel of microRNAs (miRNAs), which are promising biomarkers in many clinical fields, in patients before and after initiating ECMO. Methods Serum miRNA levels from 14 patients hospitalized for acute respiratory failure and supported with ECMO in our medical intensive care unit were analysed before and 24 hours after ECMO. In total, 179 serum-enriched miRNAs were profiled by using a real-time PCR panel. For validation, differentially expressed miRNAs were individually quantified with conventional real-time quantitative PCR at 0, 24, and 72 hours. Results Under ECMO support, platelet count significantly decreased by 65 x 10(3)/mu L (25th percentile = 154.3 x 10(3)/mu L; 75th percentile = 33 x 10(3)/mu L). Expression of the 179 miRNAs investigated in this study did not change significantly throughout the observational period. Conclusions According to our data, the expression of serum miRNAs was not altered by ECMO therapy itself. We conclude that ECMO does not limit the application of miRNAs as specific clinical biomarkers for the patients' underlying disease

Circulating HtrA2 as a novel biomarker for mitochondrial induced cardiomyocyte apoptosis and ischemia-reperfusion injury in ST-segment elevation myocardial infarction

Background: Ischemia-reperfusion (I/R) injury in ST-segment elevation myocardial infarction (STEMI) significantly contributes to overall myocardial damage. As a consequence of I/R injury in the heart, the hightemperature requirement protein A2 (HtrA2) is released from the mitochondrial intermembrane space of cardiomyocytes to the cytoplasm, whereupon it induces apoptosis. Methods: Serum was obtained from STEMI (n = 37), non-ST-segment elevation myocardial infarction (NSTEMI) (n = 20), stable coronary artery disease (CAD) (n = 17) and patients with CAD excluded (n = 9). In STEMI, I/R injury was assessed via measurement of ST-segment resolution. Results: HtrA2 was significantly increased in STEMI compared to NSTEMI, stable CAD and patients with CAD excluded (981.3 (IQR: 543.5-1526.2) pg/mL vs. 494.5 (IQR: 413.8-607) pg/mL vs. 291 (IQR: 239-458.5) pg/mL vs. 692.2 (IQR: 276.6-964.7) pg/mL; p <= 0.0001). STEMI patients with HtrA2 level of at least the median or above had a higher peak creatine kinase (CK) (p = 0.0002) and cardiac troponin T levels (cTnT) (p = 0.0019). Significantly more STEMI patients with HtrA2 levels of at least the median or above were identified as I/R injury (87% vs. 42%; p < 0.0001). Serum HtrA2 demonstrated a superior area under a curve in a receiver operating characteristic analysis for predicting I/R injury compared to CK, creatine kinase myocardial-band (CK-MB) and cTnT levels (AUC = 0.7105 vs. AUC = 0.5632 vs. AUC = 0.5660 vs. AUC = 0.5407 respectively). Conclusion: HtrA2 shows promise as a novel potential biomarker for mitochondrial-induced cardiomyocyte apoptosis and may help to identify I/R injury after STEMI. (C) 2017 Elsevier B.V. All rights reserved