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

Summary

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.

Abstract

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.

Introduction

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 linked to its capability to promote cholesterol efflux but rather to its non-cholesterol cargo, in particularly as a circulating miRNAs carrier ^{3, 4}. These miRNAs may not only modulate lipid metabolism but are also associated with anti-inflammatory, antioxidant and antithrombotic effects of the HDL-miRNA complex ^{5, 6}.

To further explore the role of miRNAs carried in HDL particles, a simple and easy protocol needs to be established for miRNA extraction from isolated highly purified HDL for use in clinical routine. Numerous methods have been described to isolate HDL. These methods are either very time consuming or require large volume of plasma that may require sample pooling, extensive dialysis for desalting isolated lipoproteins and they do not completely remove exosomes as a source of miRNAs³, respectively. Here we describe a simple and rapid method that can isolate miRNA from highly purified HDL utilizing small volume of blood samples on a larger scale. We believe that this method may serve as good reference to promote research into the role of circulating miRNAs and in particular the role of HDL in facilitating communication between various cells and organs.

Protocol

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 at 3,000 g (4 °C) in a swinging bucket rotor for 10 min to remove WBC & Platelets and then additional 15 min to remove remaining cell debris respectively.
4. Measure the density of the plasma using a densitometer at RT as per manufacture instructions.
   NOTE: Adjustment of the density (d = 1.023 g/ml) with 0.9% saline solution may be required after removal of exosomes but prior to density gradient ultracentrifugation.

2. Exosome Removal from Plasma

1. Remove the circulating exosomes that have a density similar to HDL and represent a quantitatively significant source of miRNA³.
   1. Do this by adding 252 µl exosome precipitation solution to 1 ml plasma followed by incubation for 30 min at 4 °C. To pellet out the exosomes, centrifuge the mixture for 30 min at 1,500 g at 4 °C.
   2. To isolate HDL, transfer 1 ml of the resulting supernatant to a polycarbonate thick-walled ultracentrifuge tube for further processing with density gradient ultracentrifugation (see below).

3. Density Gradient Ultracentrifugation (Figure 1).

1. To seperate HDL use a 3-step process employing a floor ultracentrifuge with a fixed-angle rotor operating at 448,811 x G and 8 °C, respectively.
2. Prepare three different density solutions sequentially and fresh for each isolation.
   1. Prepare Solution A (isolation of VLDL, d =1.006 g/ml) by dissolving 11.4 g NaCl (NaCl: 0.195 mol), 0.1 g EDTA2Na and 1 ml 1N NaOH in 1,000 ml of autoclaved-distilled water. Then add an additional 3 ml of autoclaved-distilled water.
   2. Prepare Solution B (isolation of LDL, d = 1.182 g/ml) by adding 25.2 g NaBr to 100 ml solution A (NaCl 0.195 mol, NaBr 2.44 mol).
   3. Prepare Solution C (isolation of HDL, d=1.470 g/ml) by mixing 78.8 g NaBr with 100 ml of solution A (NaCl 0.195 mol, NaBr 7.7 mol). Confirm the appropriate density at RT using a densitometer. Keep all solutions at 4 °C until further use.

4. Isolation of VLDL

1. Mix 1 ml of plasma (average density = 1.023 g/ml) and nuclease free 200 µl of Fat Red 7B in a 6.5 ml polycarbonate thick-walled ultracentrifuge tube.
2. Then carefully layer 5 ml of solution A on top of the mixture. If needed, add additional Fat Red 7B on top of solution A to balance the weight of each tube. Centrifuge for 2 hr (acceleration - 5), (deceleration - 7).
   NOTE: During centrifugation, the lipoproteins are accumulated as a band at their equilibrium density regions.
3. At the end of the run observe 2 layers. Remove 1.5 ml of the VLDL fraction representing the top layer and store at 4 °C.
4. Finally, using a pipette transfer 4 ml from the bottom of the tube containing the LDL, HDL, albumin and fatty acid fraction to a new polycarbonate tube for LDL isolation.

5. Isolation of LDL

1. Mix 2 ml of solution B and 100 µl nuclease free Fat Red 7B into the tube containing the LDL and HDL fraction (section 4), respectively.
2. Then centrifuge out for 3 hr (acceleration 9, deceleration 7). Thereafter, remove 1.5 ml of the LDL fraction representing the top layer and keep at 4 °C or store at -80 °C. Finally, transfer 4 ml from the bottom of the tube containing the HDL fraction to a new polycarbonate tube.

6. Isolation of HDL

1. Mix 2 ml of solution C, 100 µl nuclease free Fat Red 7B and 15 µl of 98% β-mercaptoethanol into the tube containing the HDL fraction, respectively.
2. Centrifuge for 3 hr (acceleration 9, deceleration 7). Thereafter remove 2 ml of the HDL fraction representing the top layer and either keep at 4 °C or store at -80 °C.

7. Desalting and Concentration of Lipoprotein Fractions

1. To avoid interference with subsequent agarose gel electrophoresis and PCR, remove excessive salt added during density gradient ultracentrifugation using centrifugal filter devices with the appropriate molecular weight cutoff (3K tube for VLDL and 10K tube for LDL/HDL) as described by the manufacturer's instructions.
   1. Briefly, after adding 2.5 ml cold PBS (137 mM NaCl, 2.7 mM KCL, 8 mM Na2HPO4, 2 mM KH2PO4; pH 7.4) centrifuge the entire VLDL fraction collected-during density gradient ultracentrifugation at 4 °C for 60 min using a swinging bucket rotor.
   2. Desalt the LDL fraction twice with 10 ml ice cold PBS for 30 min each. Next, Use 13 ml ice cold PBS twice for desalting the HDL fraction. The higher PBS volume is necessary to improve mobility with agarose gel electrophoresis. After centrifugation, remove the lipoprotein containing solutes and keep at 4 °C or stored at -80 °C.

8. Agarose Gel Electrophoresis

1. Perfrom lipoprotein agarose gel electrophoresis employing the kit with minor modifications of the manufacturer's instructions as follows.
   NOTE: This step is just to assess the quality and purity of the concentrated lipoprotein samples.
   1. Briefly, obtain 6 µl of the desalted lipoprotein fraction with density gradient ultracentrifugation and load onto a pre-cast lipoprotein gel. Use human lipoprotein standards for VLDL, LDL and HDL as size reference. Carry out electrophoresis at RT at 100 V for 60 min using Rep Prep buffer.
   2. Dry the gel for 10 min and then stain for 10 min at RT with Fat Red 7B. Destain the gel in a mixture of methanol-water 75:25 (v/v) and dry again for 5 min.

9. RNA Extraction and Purification

1. Perfrom isolation of miRNA by purified human HDL using the serum/plasma miRNA isolation and purification kit.
   1. Briefly, add 1 ml of RNA lysis reagent to 200 µl of purified HDL, mix with a vortexer and then incubated for 5 min at RT to ensure complete dissociation of nucleoprotein complexes and inactivation of RNases.
   2. Then spike 3.5 µl of synthetic Caenorhabditis elegans microRNA (cel-miR-39; 1.6 x 10⁸ copies/µl) into the mixture. Then carry out RNA extraction according to the manufacturer's instructions.
2. Perform purification of extracted-miRNA with elute spin columns as per manufacturer's instructions. Measure the concentration of miRNA from purified HDL with a spectrophorometer.
   NOTE: Elution of miRNA from the spin columns employed 16 µl of RNase-free water.

10. Reverse Transcription (RT-PCR)

1. Isolate 100 ng of the miRNA from HDL spiked with synthetic miRNA (cel-miR-39) and reverse-transcribed in a 20 µl reaction volume employing the reverse transcription kit and according to the manufacturer's instructions.
2. Perform appropriate controls without template miRNA (NTC) and without reverse transcriptase enzyme mix (NRT).

11. Real-time PCR (qRT-PCR) 

1. Perfrom Real-time PCR in a total volume of 20 µl with 2 µl of a 1:2 dilution of the cDNA, 10 µl PCR mix, 2 µl universal primer, 2 µl of miRNA primers and 4 µl RNase-free water.
2. Run the reaction in 96-well plates at 95 °C for 15 min, followed by 45 cycles of 94 °C for 15 sec and 55 °C for 30 s and an extension phase at 70 °C for 30 s.ec Perfrom all reactions in triplicates.
3. Next, Calcuate relative quantities of miRNA by using the 2^-ΔΔCt method after normalization to the synthetic housekeeping gene as per manufacturer's instructions .

Results

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

Discussion

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 ...

Materials

List of materials used in this article

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
NaOH	SIGMA-ALDRICH	480878
0.20 µM sterile syringe filter	SIGMA-ALDRICH	Z227536