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Thromb Haemost 2018; 118(07): 1257-1269
DOI: 10.1055/s-0038-1660437
New Technologies, Diagnostic Tools and Drugs
Georg Thieme Verlag KG Stuttgart · New York

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

Jose Coelho-Lima
1   Institute of Genetic Medicine, Newcastle University, International Centre for Life, Newcastle upon Tyne, United Kingdom
,
Ashfaq Mohammed
2   Department of Cardiology, Freeman Hospital, Newcastle upon Tyne NHS Foundation Trust, High Heaton, Newcastle upon Tyne, United Kingdom
,
Suzanne Cormack
2   Department of Cardiology, Freeman Hospital, Newcastle upon Tyne NHS Foundation Trust, High Heaton, Newcastle upon Tyne, United Kingdom
,
Samuel Jones
1   Institute of Genetic Medicine, Newcastle University, International Centre for Life, Newcastle upon Tyne, United Kingdom
,
Rajiv Das
2   Department of Cardiology, Freeman Hospital, Newcastle upon Tyne NHS Foundation Trust, High Heaton, Newcastle upon Tyne, United Kingdom
,
Mohaned Egred
2   Department of Cardiology, Freeman Hospital, Newcastle upon Tyne NHS Foundation Trust, High Heaton, Newcastle upon Tyne, United Kingdom
,
Pedram Panahi
2   Department of Cardiology, Freeman Hospital, Newcastle upon Tyne NHS Foundation Trust, High Heaton, Newcastle upon Tyne, United Kingdom
,
Simi Ali
3   Institute of Cellular Medicine, Newcastle University, Newcastle upon Tyne, United Kingdom
,
Ioakim Spyridopoulos
1   Institute of Genetic Medicine, Newcastle University, International Centre for Life, Newcastle upon Tyne, United Kingdom
2   Department of Cardiology, Freeman Hospital, Newcastle upon Tyne NHS Foundation Trust, High Heaton, Newcastle upon Tyne, United Kingdom
› Author Affiliations Funding Jose Coelho-Lima is supported by a CAPES Foundation PhD scholarship (Brazilian Ministry of Education; BEX 0881–14–7). 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.
Further Information

Publication History

11 January 2018

02 May 2018

Publication Date:
11 June 2018 (online)

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Abstract

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.

Keywords

microRNA - RT-qPCR normalization - heparin - bivalirudin - myocardial infarction

Supplementary Material

 

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