Establishment of a simpler method for measuring HDL-microRNAs.

BACKGROUND

MicroRNAs are present not only in exosomes but also in high-density lipoprotein (HDL) and have the potential as biomarkers for various diseases. Various purification methods have been developed to quantify HDL-miRNAs; however, they are unsuitable for clinical applications. Therefore, we aimed to establish a simpler analytical method to quantify HDL-miRNAs for clinical applications.

METHODS

We purified HDL fraction from pooled plasma using a three-step protocol consisting of ultracentrifugation, phosphotungstic acid/MgCl

RESULTS

Exosomes and LDL were not detected in the purified HDL fraction. Furthermore, we confirmed that only HDL was purified and that the HDL recovery rate of our method was at least approximately 50%. The threshold cycle values of miR-223, miR-92, miR-146a and miR-150 in the same subject were 32.11 ± 0.58, 32.50 ± 0.35, 34.30 ± 0.70 and 34.91 ± 0.77, respectively ( n = 10). The coefficient of variation values for these miRNAs were 1.08-2.21%. In addition, the standard curve for the quantitative analysis of miRNAs showed high linearity (30-30,000 copies/ μL) with a correlation coefficient of >0.99. The concentrations of HDL-miR-223 and HDL-miR-92 in the plasma of 10 subjects were 1.98 ± 0.32 and 0.90 ± 0.14 copies/mL (×10

CONCLUSIONS

We established a simple method for quantifying HDL-miRNAs and improved the sample processing capacity compared with conventional methods.