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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 SpyridopoulosORCiD



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

For re-use rights please refer to the publisher's terms and conditions.


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


DOI: 10.1055/s-0038-1660437


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Funder referenceFunder name
Ioakim Spyridopoulos is supported by grants from the British Heart Foundation as well as the Newcastle Health Care Charity and the Newcastle upon Tyne Hospitals NHS Charity.
Jose Coelho-Lima is supported by a CAPES Foundation PhD scholarship (Brazilian Ministry of Education; BEX 0881–14–7).