Establishment of a simpler method for measuring HDL-microRNAs
Hiroaki Ishikawa1, Hiroya Yamada2, Kanako Kondo3
11 Department of Biochemistry, Fujita Health University School of Health Sciences, Toyoake, Japan.
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/MgCl2 precipitation and desalting/buffer exchange followed by the quantification of HDL-miRNAs by quantitative real-time PCR. In order to establish a method to quantify HDL-miRNAs by quantitative real-time PCR, we prepared standard curves for miR-223 and miR-92. The HDL-miRNAs of 10 volunteers were assessed.
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 (×104).
Conclusions:
We established a simple method for quantifying HDL-miRNAs and improved the sample processing capacity compared with conventional methods.
Insights
Researchers developed a simpler method to quantify microRNAs in high-density lipoprotein (HDL) for clinical use. This new technique improves sample processing and aids in identifying HDL-miRNAs as disease biomarkers.
Area of Science:
- Biochemistry
- Molecular Biology
- Clinical Diagnostics
Background:
- MicroRNAs within high-density lipoprotein (HDL) show potential as disease biomarkers.
- Current methods for quantifying HDL-miRNAs are not suitable for clinical applications.
Purpose of the Study:
- To establish a simpler analytical method for quantifying HDL-miRNAs.
- To enable clinical applications of HDL-miRNA quantification.
Main Methods:
- Purification of HDL fraction from plasma using ultracentrifugation and precipitation.
- Quantification of HDL-miRNAs using quantitative real-time PCR.
- Preparation of standard curves for miR-223 and miR-92.
Main Results:
- The purification method yielded HDL without detectable exosomes or LDL, with a recovery rate of approximately 50%.
- Quantitative real-time PCR demonstrated high linearity (correlation coefficient >0.99) for miRNA quantification.
- Low coefficients of variation (1.08-2.21%) and precise quantification of specific HDL-miRNAs were achieved.
Conclusions:
- A simplified method for quantifying HDL-miRNAs has been successfully established.
- The new method offers improved sample processing capacity compared to conventional techniques.
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