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Overcoming Heparin-Associated RT-qPCR Inhibition and Normalization Issues for microRNA Quantification in Patients with Acute Myocardial Infarction

Lookup NU author(s): Dr Suzanne Cormack, Samuel Jones, Dr Rajiv Das, Dr Mohaned Egred, Professor Simi Ali, Professor Ioakim Spyridopoulos

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This is the authors' accepted manuscript of an article that has been published in its final definitive form by Georg Thieme Verlag, 2018.

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Abstract

Copyright © 2018, Schattauer GmbH. All rights reserved. Background Cardiac-enriched micro ribonucleic acids (miRNAs) are released into the circulation following ST-elevation myocardial infarction (STEMI). Lack of standardized approaches for reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) data normalization and presence of RT-qPCR inhibitors (e.g. heparin) in patient blood samples have prevented reproducible miRNA quantification in this cohort and subsequent translation of these biomarkers to clinical practice. Materials and Methods Using a RT-qPCR miRNA screening platform, we identified and validated an endogenous circulating miRNA as a normalization control. In addition, we assessed the effects of in vivo and in vitro anticoagulant drugs administration (heparin and bivalirudin) on three RT-qPCR normalization strategies (global miRNA mean, exogenous spike-in control [cel-miR-39] and endogenous miRNA control). Finally, we evaluated the effect of heparin and its in vitro inhibition with heparinase on the quantification of cardiac-enriched miRNAs in STEMI patients. Results miR-425–5p was validated as an endogenous miRNA control. Heparin administration in vitro and in vivo inhibited all RT-qPCR normalization strategies. In contrast, bivalirudin had no effects on cel-miR-39 or miR-425–5p quantification. In vitro RNA sample treatment with 0.3 U of heparinase overcame heparin-induced over-estimation of cardiac-enriched miRNA levels and improved their correlation with high-sensitivity troponin T. Conclusion miRNA quantification in STEMI patients receiving heparin is jeopardized by its effect on all RT-qPCR normalization approaches. Use of samples from bivalirudin-treated patients or in vitro treatment of heparin-contaminated samples with heparinase are suitable alternatives for miRNA quantification in this cohort. Finally, we reinforce the evidence that cardiac-enriched miRNAs early after myocardial reperfusion reflect the severity of cardiac injury.


Publication metadata

Author(s): Coelho-Lima J, Mohammed A, Cormack S, Jones S, Das R, Egred M, Panahi P, Ali S, Spyridopoulos I

Publication type: Article

Publication status: Published

Journal: Thrombosis and Haemostasis

Year: 2018

Volume: 118

Issue: 7

Pages: 1257-1269

Print publication date: 01/07/2018

Online publication date: 11/06/2018

Acceptance date: 02/05/2018

Date deposited: 03/10/2019

ISSN (print): 0340-6245

ISSN (electronic): 2567-689X

Publisher: Georg Thieme Verlag

URL: https://doi.org/10.1055/s-0038-1660437

DOI: 10.1055/s-0038-1660437


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