Investigating microRNA Biomarkers in Acute Myocardial Infarction

MicroRNAs (miRNAs) are small non-coding RNA molecules which perform a critical regulatory function in gene translation through the degradation or silencing of target mRNAs.

They are included in a varied range of biological processes such as proliferation and differentiation, hemostasis, metabolism, inflammation or apoptosis, and also in the pathophysiology of several diseases.

Many studies have proposed that circulating miRNAs are beneficial prognostic and diagnostic biomarkers of CVD.

Observing the amount of specific miRNA along with protein-based biomarkers may be a successful tool in the diagnosis of CVD and estimation of the prognosis.

In the cardiovascular system, miR-1 is highly expressed. The latest studies have shown that its expression is dysregulated in the heart under cardiovascular disease conditions, for example cardiac hypertrophy, proliferative vascular disease, and ischemic heart disease or heart failure.

miR-223 and miR-126 are included in platelet activation and endovascular inflammation, and have been referred to as biomarkers in coronary artery disease diagnosis.

The goal of the study was to explore the possible predictive value of particular miRNAs. A group of 140 patient samples with acute myocardial infarction (AMI) and 100 healthy patient samples were collected from the PRAGUE-18 study.

Method

BioVendor has recently created miREIA, a novel technique for the quantification of miRNA, based on the enzyme immunoassay format.

This unique approach entails the hybridization of miRNA isolated from the sample of a patient to a complementary biotinylated DNA oligonucleotide probe.

The DNA/RNA hybrids are then moved onto a stationary solid phase covered with a monoclonal antibody that is specific to the ideally matched DNA/miRNA hybrids.

The solid phase is incubated with streptavidin-HRP conjugate post-washing and the subsequent complexes are visualized with a chromogenic substrate, after a further washing step.

Four miREIAs were produced to evaluate miRNAs (miR-1-3p, miR-126-3p, miR-223-3p and cel-miR-39-3p) isolated from the whole blood. The standard analytical features (miR-223-3p assay) were: LOD = 0.13 amol/µl, intraassay CV 8%, dilution recovery 102%, spiking recovery 98%.

miRNA Quantification

Image Credit: BioVendor

A. Hybridization of miRNA to Complementary Biotinylated DNA.

A predetermined amount of specific biotin-labeled DNA oligonucleotide is hybridized to miRNA isolated from the sample of blood.

B. miREIA – miRNA Enzyme Immunoassay

The mixture for hybridization is moved onto a stationary solid phase covered with a monoclonal antibody particular to the perfectly matched RNA/DNA-biotin hybrids.

In the next stage, the solid phase is washed and then incubated with the streptavidin-HRP conjugate. Lastly, the subsequent complexes are visualized by chromogenic substrate 3,3’,5,5’-tetramethylbenzidine (TMB).

miREIA-223-3p

C. Calibration Range

Image Credit: BioVendor

The miREIA-223-3p assay’s calibration range is 0.39 to 12.5 amol/ul.

D. Dilution Linearity

Taken from whole blood samples, two microRNA isolates were serially diluted.

Source: BioVendor

Sample Sample dilution Observed concentration amol/µl Expected concentration amol/µl Recovery O/E (%)
Sample 1 - 363.4 - -
2 174.5 181.7 96.0
4 94.2 90.8 103.7
8 46.6 45.4 102.5
Sample 2 - 531.3 - -
2 279.8 265.7 105.3
4 140.2 132.8 105.5
8 64.3 66.4 96.8

 

The recovery was calculated to be between 96 and 106%.

E. Comparison of Methods – miREIA Versus qRT-PCR

Synthetic non-human cel-miR-39-3p was introduced to 139 whole blood samples throughout the RNA isolation process.

After RNA isolation, the concentration of exogenous cel-miR-39-3p was quantified by employing the qRT-PCR (TaqMan Advanced miRNA Assay, ThermoFisher) and the immunoassay cel-miR-39-3p miREIA (BioVendor).

Image Credit: BioVendor

A significant correlation was found between the concentration of cel-miR39-3p identified by miREIA and qRT-PCR, the Pearson correlation coefficient was more than 0.68.

F. Measurement of miR-223 in Patient with AMI and Control Group

RNA was isolated from 89 whole blood samples from patients without acute myocardial infarction and 139 whole blood samples of patients with acute myocardial infarction.

The concentration of miR223-3p was quantified by employing miREIA kit. The reported concentrations of miR-223-3p were normalized by miR-126-3p. The normalized concentration of miR223-3p in patients with AMI was extensively higher when compared to the control group (p<0,000001).

Image Credit: BioVendor

Image Credit: BioVendor

The receiver operating characteristic (ROC) curves were constructed to analyze the diagnostic potential of normalized miR-223-3p.

The ROC curves similarly demonstrated high separation between the groups with and without acute myocardial infarction, with an AUC of 0.76, a specificity of 71.9 and a sensitivity of 76.3.

Conclusion

  • BioVendor is launching a novel approach for the absolute quantification of microRNA in clinical samples with no need for amplification and reverse transcription steps.
  • The miREIA technique offers successful analytical features and a strong correlation with the qRT-PCR technique.
  • This technique provides further opportunities to evaluate miRNA utilizing traditional immunoassay equipment that is widely available, promoting the efficient adoption of miRNA biomarkers in the laboratory and clinical practice.

References and Further Reading

  1. Kappel, Andreas, et al. MicroRNA in vitro diagnostics using immunoassay analyzers. Clinical chemistry 61.4 (2015): 600-607.
  2. Romaine, Simon PR, et al. MicroRNAs in cardiovascular disease: an introduction for clinicians. Heart 101.12 (2015): 921-928.
  3. Wang, Jin, Jinyun Chen, and Subrata Sen. MicroRNA as biomarkers and diagnostics. Journal of cellular physiology 231.1 (2016): 25-30.
  4. Chen, Yajing, et al. Increased circulating exosomal miRNA-223 is associated with acute ischemic stroke. Frontiers in neurology 8 (2017): 57.
  5. Taïbi, Fatiha, et al. miR-223: an inflammatory oncomiR enters the cardiovascular field. Biochimica et Biophysica Acta (BBA)-Molecular Basis of Disease 1842.7 (2014): 1001-1009.
  6. Motovska, Zuzana, et al. Prasugrel Versus Ticagrelor in Patients With Acute Myocardial Infarction Treated With Primary Percutaneous Coronary Intervention Clinical Perspective: Multicenter Randomized PRAGUE-18 Study. Circulation 134.21 (2016): 1603-1612.

Acknowledgments

Produced from materials originally authored by J. Izáková1, M. Hlozankova1, B. Dvorakova1, M. Buresova1, M. Holcapkova1, L.Chalupova1, K. Cuchnova1, M. Karpisek1,4, T. Mrackova1, E. Bace1, Z. Motovska2, M. Hromadka3, PRAGUE-18 Study Group, and V. Ruzicka1 from Biovendor.

  1. BioVendor Laboratorní medicína a.s., Research and Diagnostic Division, Brno, Czech Republic
  2. Cardio center, Third Faculty of Medicine, Charles University and University Hospital Kralovske Vinohrady, Prague, Czech Republic
  3. Cardiology Department, University Hospital and Faculty of Medicine in Pilsen and Faculty Hospital, Charles University, Czech Republic
  4. Department of Human Pharmacology and Toxicology, University of Veterinary and Pharmaceutical Sciences, Brno, Czech Republic

About BioVendor

BioVendor Group is an international diagnostics company with its headquarters in Brno, Czech Republic.

BioVendor provides a broad range of IVD technologies and capabilities including, in-house development and manufacturing of immunoassays and molecular diagnostics for the clinical market, as well as a wide offering for the Life Sciences market.

The core of the portfolio consists of immunoassays in several formats, both single-test and multiplexed. The portfolio is focused on infectious serology, autoimmunity, endocrinology, and newly-discovered biomarkers.

Since its establishment in 1992, BioVendor has placed high importance on innovation and value brought to customers via providing diagnostics solutions. Recent examples include, fully automated ELISA platform, proprietary microarray solution, Next Generation Sequencing kits, and proprietary miRNA immunoassays.

BioVendor is committed to its strategy to grow internationally. BioVendor has an extensive track record of acquisitions including, TestLine Clinical Diagnostics (Czech Republic), ViennaLab Diagnostics (Austria), Oxford Biosystems (UK), and most recently in September 2017, DIAsource Immuno-Assays (Belgium).


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Last updated: Nov 18, 2019 at 5:21 AM

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