Heparin selectively affects the quantification of microRNAs in human blood samples.

MicroRNAs (miRNAs) are noncoding RNA molecules that either inhibit translational processing or mediate degradation of target mRNAs in various physiological and pathophysiological processes (1 ). miRNAs have been detected in several body fluids, such as blood plasma, and have emerged as potentially suitable biomarkers for various diseases, including cancer and myocardial infarction (2 ). Heparin is commonly used as an anticoagulant in cardiovascular diagnostics and interventions. Previous studies have demonstrated that heparin can inhibit RNA quantification in vitro by interfering with the DNA polymerase used in the quantitative PCR (qPCR) reaction (3 ). It is unclear, however, whether the biologically active concentrations of heparin achieved after treatment of patients influences the quantification of miRNAs in blood samples. We therefore assessed the effect of systemic application of heparin on the measurement of endogenous circulating miRNAs in plasma samples. Arterial blood samples from 11 patients were obtained from the femoral artery before the cardiac catheterization procedure and at 10 and 60 min after heparin was administered (bolus, 5000 IU heparin; maintenance dose, 2500 – 5000 IU). We isolated RNA from EDTA-treated plasma with a TRIzol-based miRNA-isolation protocol (miRNeasy; Qiagen). We used a hydrolysis probe– based quantitative PCR method to measure several vascular-related miRNAs (miR-17, miR-34a, miR92a, miR-126, miR-145, and miR378). We also measured miR-1, miR-133a, miR-208, and miR-499, which have been suggested as biomarkers of acute myocardial infarction. The concentrations of miR-34a, miR-133a, miR-208, miR-378, and miR-499 were significantly and profoundly reduced from baseline by 10 min after heparin administration (P 0.05) and remained reduced for 60 min, although only miR-34a, miR-133a, and miR-208 achieved statistically significant reductions at 60 min (P 0.05) (Fig. 1). Furthermore, the relative concentration of circulating miR-34a was inversely correlated with the activated clotting time (r 0.69; P 0.01). Normalizing miRNA production in plasma is challenging because of the lack of a validated control miRNA that is not regulated. Therefore, we supplemented plasma samples with Caenorhabditis elegans miR-39 (cel-miR-39), as described previously (4 ). Surprisingly, the recombinant cel-miR39, which we spiked into plasma samples after adding TRIzol to normalize RNA extractions, had decreased profoundly and significantly at 10 min (P 0.001) and remained decreased by 30% after 60 min (P 0.11). Next, we determined whether the detection of all recombinant miRNAs spiked into plasma samples is affected by heparin in the plasma. miR-499 detection was again suppressed significantly when it was added to samples obtained at 10 min [mean (SD), 65% (18%); P 0.001)] or at 60 min (P 0.006) after heparin administration. Furthermore, miR-499 and cel-miR-39 concentrations were inversely correlated with the activated clotting time [r 0.68 (P 0.02), and r 0.68 (P 0.01), respectively]. We observed no significant interference with PCR-based detection, however, when recombinant miR126 and miR-92a were spiked into plasma samples from patients who received heparin (all P values 0.05), suggesting that the presence of biologically active heparin influences the detection of only specific miRNAs measured by the PCR in vitro. To determine whether heparin might interfere directly and selectively with the detection of specific miRNAs via qPCR assays, we measured recombinant miRNAs by qPCR in the presence of increasing concentrations of heparin. Heparin dose-dependently inhib1 Nonstandard abbreviations: miRNA, microRNA; qPCR, quantitative qPCR; cel-miR-39, Caenorhabditis elegans miR-39. 1.8 celmiR-39 miR-17 miR-126 miR-145 miR-499 miR-378 miR-34a miR-133a miR-1 Vascular-related miRNAs Cardiac biomarker