Método rápido y simplificado para el aislamiento alto rendimiento de procesamiento de miRNA desde muy puras lipoproteína de alta densidad
Summary
MicroARNs juegan un importante papel regulador y están surgiendo como nuevas dianas terapéuticas para diversas enfermedades humanas. Se ha demostrado que miRNAs se realizan en las lipoproteínas de alta densidad. Hemos desarrollado un método simplificado para aislar rápidamente HDL purificado adecuado para el análisis de los genes miARN partir de plasma humano.
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
Representative Results
Discussion
Identificación de nuevos biomarcadores de la sangre será ayudar en el diagnóstico clínico y el pronóstico de diversas enfermedades. Los microARN han sabe que poseen todas las cualidades de biomarcadores y se ha demostrado en diversos estudios 14-17. En este estudio hemos demostrado método fácil rápido y sencillo para aislar los genes miARN de HDL plasma. Densidad convencional gradiente método de ultra-centrifugación de aislamiento de VLDL, LDL y HDL depende de muestreo preciso de plasma, una prepara…
Disclosures
The authors have nothing to disclose.
Acknowledgements
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.
Materials
| 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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