Search

Heparin, a widely used anticoagulant in cardiovascular diseases, is notorious for its inhibitory effect on qRT-PCR-based detection. Heparinase I could degrade heparin in RNA. qRT-PCR-based TaqMan Low Density Array (TLDA) technology is commonly used for circulating microRNAs (miRNAs) profiling analysis. However, the effect of heparin contamination on inhibition of miRNAs TLDA amplification, as well as the method for removing heparin during this process, are not yet well investigated. We obtained the plasma RNA samples from patients undergoing percutaneous coronary intervention (PCI) before and after heparinization (n = 26). We found that heparin suppressed the miRNAs amplification by ∼8 cycles in the TLDA assay, which was absolutely reversed after treating the RNA samples with heparinase I using the components from TLDA reverse transcription system. We further observed that heparin inhibited the miRNAs amplification by ∼4 cycles in the qRT-PCR assay, which was also reversed by heparinase I using the similar method. Furthermore, we demonstrated that plasma miR-92a and miR-155 were differentially expressed in the patients undergoing PCI tested by TLDA assay, which was validated by qRT-PCR. In conclusion, we present a simple method for the removal of heparin with heparinase I, and for the subsequent successful miRNAs TLDA or RT-qPCR amplification. [ABSTRACT FROM AUTHOR]

The activation of a hemostatic system plays a critical role in the incidence of acute coronary events. Hemostatic proteins may be regulated by microRNAs (miRNAs). Microparticles (MPs) are the major carrier of circulating miRNAs. The aim of this study was to determine the potential role of miRNAs in regulating gene expression involved in the hemostatic system in patients with unstable angina (UA). MiRNA expression profiles in the plasma from patients with UA (UA group, n = 9) compared with individuals with clinical suspicion of coronary artery disease (CAD) but negative angiography (control group, n = 9) showed that among 36 differentially expressed miRNAs, miR-19b was the most obvious one. Using real-time PCR, 5 selected miRNA levels in plasma (UA group, n = 20; control group, n = 30) and plasma MPs (UA group n = 6; control group n = 6) were proved to be consistent with the miRNA array. Flow cytometry analysis indicated that the amounts of plasma endothelial microparticles (EMPs) were increased in UA patients (UA group, n = 4) compared to controls (control group, n = 4). In cultured endothelial cells (ECs), TNF-α increased miR-19b release and expression. Tissue factor (TF) was predicted to be the target of miR-19b by bioinformatics analysis. Luciferase reporter assays demonstrated that miR-19b binds to TF mRNA. Overexpression of miR-19b inhibited TF expression and procoagulant activity. This study indicates that in UA patients, the increase of miR-19b wrapped in EMPs due to endothelial dysfunction may partially contribute to the circulating miR-19b elevation and miR-19b may play an anti-thrombotic role by inhibiting the expression of TF in ECs. [ABSTRACT FROM AUTHOR]