Elsevier

International Journal of Cardiology

Volume 257, 15 April 2018, Pages 247-254
International Journal of Cardiology

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

https://doi.org/10.1016/j.ijcard.2017.10.111Get rights and content

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. 484.7 copies/μl; p = 0.0004 for ddPCR and 136.4 vs. 122.8 copies/μl; p = 0.2273 for qRT-PCR) and in miR-208a (0 vs. 24.1 copies/μl, p = 0.0013 for ddPCR and 0 vs. 0 copies/μl, p = 0.0032 for qRT-PCR), with similar differences observed in miR-499 levels (9.4 vs. 81.5 copies/μl, p < 0.0001 for ddPCR and 0 vs. 19.41 copies/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.

Introduction

Ischemic heart disease is the leading cause of morbidity and mortality worldwide [1]. Early diagnosis and intervention is integral in reducing injury in acute myocardial infarction (AMI). The current gold standard for diagnosis uses high-sensitivity cardiac troponins (hs-cTn), however these markers are not without their limitations. hs-cTn have compromised specificity, with an increased detection of no-ischemic damage [2], [3], [4]. Moreover, raised troponins are not entirely AMI specific, and they can be chronically raised in patients with congestive cardiac failure or chronic kidney disease [2], [3], [4], [5]. The establishment of novel biomarkers for AMI has therefore become an important focus of medical research. Furthermore, there is a relative paucity of established biomarkers for reperfusion injury. In recent years miRNAs have been identified as promising markers for a number of diseases, including AMI [6]. miRNAs are short (~ 22 nucleotide) non-coding single-stranded RNA molecules involved in the regulation of messenger RNA (mRNA) via inhibitory effects on translation and/or stability, ultimately influencing gene expression [7]. Under physiological or pathological conditions, miRNAs can be released from their cells of origin into the circulation and are thought to act upon cells and tissues at distant sites [8]. miRNAs are attractive candidates for disease biomarkers. They are highly stable in the circulation and measureable in a variety of bodily fluids [7], [8]. Moreover, their levels can change significantly in pathological states, and some miRNAs show high tissue and disease specificity [7]. A number of miRNAs have been investigated as valid biomarkers in AMI. Of particular interest for this study are miRNA-21, miRNA-208a and miRNA-499. These miRNAs have been shown to be upregulated in AMI, and miRNA-499 has even shown potential as a biomarker for reperfusion injury [9], [10], [11], [12], [13], [14], [15]. Although results are encouraging, a variety of issues have arisen which have limited their use [16]. Foremost amongst these limitations is the current method used to quantify miRNAs, qRT-PCR [17]. ddPCR is a relatively novel method of PCR, which partitions a 20 μl sample into ~ 20,000 individual droplets [18]. The ddPCR system uses a Poisson statistical analysis of fluorescent signals from positive and negative droplets to allow for absolute quantification [18]. ddPCR has demonstrated a number of advantages over conventional qRT-PCR, which may aid in mitigating the current limitations on using circulating miRNAs as biomarkers. In experiments to date, ddPCR has demonstrated a high degree of linearity and quantitative correlation in measuring miRNAs within its dynamic range, and has shown greater reproducibility and less inter- and intra-assay variability compared to qRT-PCR [18], [19]. Through these advantages ddPCR could offer greater day-to-day comparability and reliability and hence greater utility as both a diagnostic method as well as in validating miRNAs in large multi-center clinical trials. The aim of this study was to investigate ddPCR as a novel method of quantifying serum miRNAs and to determine whether its use leads to an improvement in the diagnostic potential of validated miRNAs for AMI and I/R injury.

Section snippets

Synthetic oligonucleotide dilution series of C. elegans-miR-39

A lyophilised 5′-phosphorylated synthetic oligonucleotide for Caenorhabditis elegans-miR-39 (C. elegans-miR-39) (Integrated DNA Technologies) (Table A.1), with a known starting mass of 7.6 nmol was centrifuged according to the company's protocol and diluted in molecular grade Tris-EDTA (TE) buffer (Thermo Fisher Scientific) to a final concentration of 10 pmol/μl.

A 12-step dilution series using nuclease-free water (Applichem Panreac) from 2500 copies/μl to 0 copies/μl was performed for

Dilution series of synthetic C. elegans-miR-39

ddPCR consistently exhibited a superior coefficient of variation (CV%) between most dilution points across all four runs (Figs. 1a/b and A.2). qRT-PCR had an average CV% of 32.9%, whilst ddPCR's average CV% was 12.1%. ddPCR demonstrated an average decrease in CV% of 50.3%. ddPCR also demonstrated superior linearity across most runs (Fig. A.3), with an average r2 of 0.983 compared to 0.945 for qRT-PCR. Finally, ddPCR demonstrated superior or equal LOD, LOQ and LLLR across all runs (Fig. 1c). It

Discussion

Droplet digital PCR (ddPCR) is a relatively novel method of PCR that allows absolute quantification of nucleic acids. With the exception of Hindson et al., at current there has been limited experimentation comparing ddPCR to qRT-PCR for quantifying miRNAs [19]. Furthermore, to the best of our knowledge, there has been negligible experimentation of ddPCR and its utility for diagnosing AMI or reperfusion injury with miRNAs. With this report, we aim to demonstrate these proposed advantages of

Limitations

The results of this study support our hypotheses. However, our study has some limitations. The clinical characteristics were obtained retrospectively and hence we were not able to collect certain data and our results may be subject to bias and confounders. Furthermore, the clinical data is limited by lack of randomization and blinding. This study was also limited by its small patient size as well as the small number of miRNAs analyzed. A potential strength is that blood for miRNA analysis was

Conclusions

In conclusion, we have shown that ddPCR consistently demonstrated superiority in both technical proficiency and diagnostic potential compared to qRT-PCR. However, further investigation is required in larger patient cohorts. Ultimately, our findings support the use of ddPCR over qRT-PCR for more accurate and reproducible quantification of miRNAs in biological expressions in cardiovascular biology, particularly for the use in large multi-center clinical trials.

The following are the supplementary

Funding sources/disclosures

The authors have no relevant financial information or potential conflicts of interest to disclose.

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    1

    This author takes responsibility for all aspects of the reliability and freedom from bias of the data presented and their discussed interpretation.

    2

    Drs Hortmann and Ahrens contributed equally to this article.

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