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Fast and Simplified Method for High Through-put Isolation of miRNA from Highly Purified High Density Lipoprotein

Published: July 27th, 2016



1Division of Gastroenterology, Hepatology and Nutrition, Virginia Commonwealth University, 2McGuire Veterans Affairs Medical Center, Research BC-101

MicroRNAs play an important regulatory role and are emerging as novel therapeutic targets for various human diseases. It has been shown that miRNAs are carried in high density lipoproteins. We have developed a simplified method to rapidly isolate purified HDL suitable for miRNA analysis from human plasma.

Small non-coding RNAs (miRNAs) have been implicated in a variety of human diseases including metabolic syndromes. They may be utilized as biomarkers for diagnosis and prognosis or may serve as targets for drug development, respectively. Recently it has been shown that miRNAs are carried in lipoproteins, particularly high density lipoproteins (HDL) and are delivered to recipient cells for uptake. This raises the possibility that miRNAs play a critical and pivotal role in cellular and organ function via regulation of gene expression as well as messenger for cell-cell communications and crosstalk between organs. Current methods for miRNA isolation from purified HDL are impractical when utilizing small samples on a large scale. This is largely due to the time consuming and laborious methods used for lipoprotein isolation. We have developed a simplified approach to rapidly isolate purified HDL suitable for miRNA analysis from plasma samples. This method should facilitate investigations into the role of miRNAs in health and disease and in particular provide new insights into the variety of biological functions, outside of the reverse cholesterol transport, that have been ascribed to HDL. Also, the miRNA species which are present in HDL can provide valuable information of clinical biomarkers for diagnosis of various diseases.

MicroRNAs are endogenous non-coding tiny RNA species that are highly conserved and are considered key players in the regulation of various biological processes by degrading or repressing specific target messenger RNAs1. Because miRNAs act intracellularly they have been explored as tissue-derived biomarkers which led to the discovery of tissue-specific functions of these miRNA. However, miRNAs are also found extracellularly either associated with proteins or in exosomes/micro vesicles that effectively can shield them from degradation by extracellular RNases2. More recent studies have shown that the protective effect of HDL may not be closely linke....

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1. Collection of Blood Samples

  1. Collect fasting peripheral venous blood samples into 10 ml plastic tubes containing anticoagulant Ethylenediaminetetraacetic acid (EDTA) (which has several advantages over other anticoagulants) by standard venipuncture of a prominent vein in the antecubital fossa.
  2. Centrifuge the blood samples at 1,600 x g for 20 min at 4 °C in a tabletop centrifuge to obtain plasma free of red blood cells and small amounts of RNA.
  3. Sequentially centrifuge the supernatant .......

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Isolation of High Density Lipoprotein After Removal of Exosomes
To obtain miRNA from highly purified HDL it is necessary to remove exosomes that represent a source of miRNA contamination7. This was done prior to density gradient ultracentrifugation with a commercially available kit. For practical purposes a three step standard density gradient ultracentrifugation protocol developed by commercial company was modified (Figure 1

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Identification of novel biomarkers from blood will aid in the clinical diagnosis and prognosis of various diseases. MicroRNAs have known to possess all the qualities of biomarkers and have been shown in various studies 14-17. In this study we have demonstrated rapid and simple easy method to isolate miRNA from plasma HDL. Conventional density gradient ultra-centrifugation method of isolation of VLDL, LDL and HDL depends on accurate sampling of plasma, precise preparation of the buffer solution, measurement of .......

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This work was supported, in whole or in part, by NIH Grants R01 AA 020758-04, U01DK 061731-13 and T32 DK 007150-38 to AJS and T32 DK 007150-38 to AA. This is original work and is not under consideration elsewhere for publication.


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Name Company Catalog Number Comments
Plastic Vacutainer Lavender K2EDTA tubes  Becton, Dickinson and Company 366643
Centrifuge Thermo Scientific, Sorvall Legend X1R  75004261
Densito 30PX densitometer Mettler Toledo MT51324450
ExoQuick solution  Invitrogen 4484451
Polycarbonate thick-walled ultracentrifuge tube Thermo Scientific O3237
Sorvall WX100 ultracentrifuge  Thermo Scientific 46902
Fat Red 7B  Sigma-Aldrich 201618
β-mercaptoethanol  Sigma-Aldrich
Amicon Ultra-15 Centrifugal filter devices 10K Millipore UFC901008
Amicon Ultra-centrifugal filter devices 3K Millipore UFC800308
QuickGel Lipo kit  Helena Laboratories  3344,3544T
Human lipoprotein standards for VLDL, LDL and HDL LipoTrol; Helena Laboratories 5069
Rep Prep buffer  Helena Laboratories  3100
RNeasy MinElute spin columns  Qiagen
NanoDrop 1000 analyzer Thermo Scientific
miScript II RT Kit  Qiagen 218161
CFX96 Touch real-time PCR detection system BioRad
miRNeasy Serum/Plasma Kit QIAGEN 217184
miScript Primer Assays QIAGEN 141078139
miScript SYBR Green PCR Kit  QIAGEN 218073
miRNeasy Serum/Plasma Spike-In Control QIAGEN 219610

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