Establishment of a simpler method for measuring HDL-microRNAs

Hiroaki Ishikawa, Hiroya Yamada, Kanako Kondo, Takeru Ota, Mirai Yamazaki, Yoshitaka Ando, Genki Mizuno, Eiji Munetsuna, Koji Suzuki, Ryoji Teradaira, Koji Ohashi

Research output: Contribution to journalArticle

1 Citation (Scopus)

Abstract

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 2 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 (×10 4 ). Conclusions: We established a simple method for quantifying HDL-miRNAs and improved the sample processing capacity compared with conventional methods.

Original languageEnglish
Pages (from-to)49-55
Number of pages7
JournalAnnals of Clinical Biochemistry
Volume56
Issue number1
DOIs
Publication statusPublished - 01-01-2019

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HDL Lipoproteins
MicroRNAs
Exosomes
Real-Time Polymerase Chain Reaction
Phosphotungstic Acid
Plasmas
Salt removal
Ultracentrifugation
Biomarkers
Purification
Volunteers
Ion exchange
Buffers

All Science Journal Classification (ASJC) codes

  • Clinical Biochemistry

Cite this

Ishikawa, Hiroaki ; Yamada, Hiroya ; Kondo, Kanako ; Ota, Takeru ; Yamazaki, Mirai ; Ando, Yoshitaka ; Mizuno, Genki ; Munetsuna, Eiji ; Suzuki, Koji ; Teradaira, Ryoji ; Ohashi, Koji. / Establishment of a simpler method for measuring HDL-microRNAs. In: Annals of Clinical Biochemistry. 2019 ; Vol. 56, No. 1. pp. 49-55.
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abstract = "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 2 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 (×10 4 ). Conclusions: We established a simple method for quantifying HDL-miRNAs and improved the sample processing capacity compared with conventional methods.",
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Establishment of a simpler method for measuring HDL-microRNAs. / Ishikawa, Hiroaki; Yamada, Hiroya; Kondo, Kanako; Ota, Takeru; Yamazaki, Mirai; Ando, Yoshitaka; Mizuno, Genki; Munetsuna, Eiji; Suzuki, Koji; Teradaira, Ryoji; Ohashi, Koji.

In: Annals of Clinical Biochemistry, Vol. 56, No. 1, 01.01.2019, p. 49-55.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Establishment of a simpler method for measuring HDL-microRNAs

AU - Ishikawa, Hiroaki

AU - Yamada, Hiroya

AU - Kondo, Kanako

AU - Ota, Takeru

AU - Yamazaki, Mirai

AU - Ando, Yoshitaka

AU - Mizuno, Genki

AU - Munetsuna, Eiji

AU - Suzuki, Koji

AU - Teradaira, Ryoji

AU - Ohashi, Koji

PY - 2019/1/1

Y1 - 2019/1/1

N2 - 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 2 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 (×10 4 ). Conclusions: We established a simple method for quantifying HDL-miRNAs and improved the sample processing capacity compared with conventional methods.

AB - 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 2 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 (×10 4 ). Conclusions: We established a simple method for quantifying HDL-miRNAs and improved the sample processing capacity compared with conventional methods.

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