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
