Droplet digital PCR as a novel detection method for quantifying microRNAs in acute myocardial infarction
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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2019, Biochemical and Biophysical Research CommunicationsCitation Excerpt :Using bioinformatics analysis (starBase) [29], we found FTX could directly target miR-21-5p with molecular binding at 3′-UTR and luciferase reporter assay validated the prediction. As previously described, miR-21 is mainly involved in transcriptional and post-transcriptional regulation and regulating cell development processes [30–32]. It has been the first miRNA found to be associated with human glioblastoma multiform [33] and deregulation of miR-21-5p was related with initiation and progression of various malignancies such as lung [34], colon [35], and bladder cancer [36].
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2019, NeuropharmacologyCitation Excerpt :Therefore, these factors must be considered in the design of studies and the inclusion of appropriate controls when investigating candidate miRNA biomarkers. Nevertheless, the potential to apply highly sensitive and quantitative methods such as RNAseq and droplet digital PCR (ddPCR) for identification, verification, and validation can significantly improve the sensitivity and specificity of potential circulating miRNA biomarkers when compared to proteins (Robinson et al., 2018). Although CSF may serves as an ideal source for miRNA due to its direct contact with the brain, blood collection is obviously less invasive and affords the collection of larger volumes (Stoicea et al., 2016).
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2019, International Journal of CardiologyCitation Excerpt :In contrast, another study found that none of the tested miRNAs outperformed cTn [36]. Digital polymerase chain reaction has been shown to exhibit superior technical qualities (decreased variability, increased day-to-day reproducibility and superior sensitivity) for quantifying miRNA concentrations in the circulation [37–39]. Droplet digital polymerase chain reaction (ddPCR) is an end-point analysis, allowing direct absolute quantification of microRNAs by using a Poisson statistical analysis of fluorescent signals from positive and negative droplets [37].
- 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.