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.

1. Collection of Blood Samples
<ol>
	<li>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.</li>
	<li>Centrifuge the blood samples at 1,600 x g for 20 min at 4 &#176;C in a tabletop centrifuge to obtain plasma free of red blood cells and small amounts of RNA.</li>
	<li>Sequentially centrifuge the supernatant at 3,000 g (4 &#176;C) in a swinging bucket rotor for 10 min to remove WBC &#38; Platelets and then additional 15 min to remove remaining cell debris respectively.</li>
	<li>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.</li>
</ol>
2. Exosome Removal from Plasma
<ol>
	<li>Remove the circulating exosomes that have a density similar to HDL and represent a quantitatively significant source of miRNA3.
	<ol>
		<li>Do this by adding 252 &#181;l exosome precipitation solution to 1 ml plasma followed by incubation for 30 min at 4 &#176;C. To pellet out the exosomes, centrifuge the mixture for 30 min at 1,500 g at 4 &#176;C.</li>
		<li>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).</li>
	</ol>
	</li>
</ol>
3. Density Gradient Ultracentrifugation (Figure 1).
<ol>
	<li>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 &#176;C, respectively.</li>
	<li>Prepare three different density solutions sequentially and fresh for each isolation.
	<ol>
		<li>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.</li>
		<li>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).</li>
		<li>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 &#176;C until further use.</li>
	</ol>
	</li>
</ol>
4. Isolation of VLDL
<ol>
	<li>Mix 1 ml of plasma (average density = 1.023 g/ml) and nuclease free 200 &#181;l of Fat Red 7B in a 6.5 ml polycarbonate thick-walled ultracentrifuge tube.</li>
	<li>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.</li>
	<li>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 &#176;C.</li>
	<li>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.</li>
</ol>
5. Isolation of LDL
<ol>
	<li>Mix 2 ml of solution B and 100 &#181;l nuclease free Fat Red 7B into the tube containing the LDL and HDL fraction (section 4), respectively.</li>
	<li>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 &#176;C or store at -80 &#176;C. Finally, transfer 4 ml from the bottom of the tube containing the HDL fraction to a new polycarbonate tube.</li>
</ol>
6. Isolation of HDL
<ol>
	<li>Mix 2 ml of solution C, 100 &#181;l nuclease free Fat Red 7B and 15 &#181;l of 98% &#946;-mercaptoethanol into the tube containing the HDL fraction, respectively.</li>
	<li>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 &#176;C or store at -80 &#176;C.</li>
</ol>
7. Desalting and Concentration of Lipoprotein Fractions
<ol>
	<li>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&#39;s instructions.
	<ol>
		<li>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 &#176;C for 60 min using a swinging bucket rotor.</li>
		<li>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 &#176;C or stored at -80 &#176;C.</li>
	</ol>
	</li>
</ol>
8. Agarose Gel Electrophoresis
<ol>
	<li>Perfrom lipoprotein agarose gel electrophoresis employing the kit with minor modifications of the manufacturer&#39;s instructions as follows. 
	NOTE: This step is just to assess the quality and purity of the concentrated lipoprotein samples.
	<ol>
		<li>Briefly, obtain 6 &#181;l 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.</li>
		<li>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.</li>
	</ol>
	</li>
</ol>
9. RNA Extraction and Purification
<ol>
	<li>Perfrom isolation of miRNA by purified human HDL using the serum/plasma miRNA isolation and purification kit.
	<ol>
		<li>Briefly, add 1 ml of RNA lysis reagent to 200 &#181;l 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.</li>
		<li>Then spike 3.5 &#181;l of synthetic Caenorhabditis elegans microRNA (cel-miR-39; 1.6 x 108 copies/&#181;l) into the mixture. Then carry out RNA extraction according to the manufacturer&#39;s instructions.</li>
	</ol>
	</li>
	<li>Perform purification of extracted-miRNA with elute spin columns as per manufacturer&#39;s instructions. Measure the concentration of miRNA from purified HDL with a spectrophorometer. 
	NOTE: Elution of miRNA from the spin columns employed 16 &#181;l of RNase-free water.</li>
</ol>
10. Reverse Transcription (RT-PCR)
<ol>
	<li>Isolate 100 ng of the miRNA from HDL spiked with synthetic miRNA (cel-miR-39) and reverse-transcribed in a 20 &#181;l reaction volume employing the reverse transcription kit and according to the manufacturer&#39;s instructions.</li>
	<li>Perform appropriate controls without template miRNA (NTC) and without reverse transcriptase enzyme mix (NRT).</li>
</ol>
11. Real-time PCR (qRT-PCR)&#160;
<ol>
	<li>Perfrom Real-time PCR in a total volume of 20 &#181;l with 2 &#181;l of a 1:2 dilution of the cDNA, 10 &#181;l PCR mix, 2 &#181;l universal primer, 2 &#181;l of miRNA primers and 4 &#181;l RNase-free water.</li>
	<li>Run the reaction in 96-well plates at 95 &#176;C for 15 min, followed by 45 cycles of 94 &#176;C for 15 sec and 55 &#176;C for 30 s and an extension phase at 70 &#176;C for 30 s.ec Perfrom all reactions in triplicates.</li>
	<li>Next, Calcuate relative quantities of miRNA by using the 2-&#916;&#916;Ct method after normalization to the synthetic housekeeping gene as per manufacturer&#39;s instructions .</li>
</ol>

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C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNAs+are+transported+in+plasma+and+delivered+to+recipient+cells+by+high-density+lipoproteins.">MicroRNAs are transported in plasma and delivered to recipient cells by high-density lipoproteins.</a> Nat Cell Biol. 13 (4), 423-433 (2011).</li><li>Wagner, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+levels+and+cellular+transfer+of+circulating+lipoprotein-bound+microRNAs.">Characterization of levels and cellular transfer of circulating lipoprotein-bound microRNAs.</a> Arterioscler Thromb Vasc Biol. 33, 1392-1400 (2013).</li><li>Wang, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNAs+185,+96,+and+223+repress+selective+high-density+lipoprotein+cholesterol+uptake+through+posttranscriptional+inhibition.">MicroRNAs 185, 96, and 223 repress selective high-density lipoprotein cholesterol uptake through posttranscriptional inhibition.</a> Mol Cell Biol. 33 (10), 1956-1964 (2013).</li><li>Rayner, K. 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The authors have nothing to disclose.

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 ...

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.

<table><tbody><tr><td>Plastic Vacutainer Lavender K2EDTA tubes&amp;nbsp;</td><td>Becton, Dickinson and Company</td><td>366643</td><td></td></tr><tr><td>Centrifuge</td><td>Thermo Scientific, Sorvall Legend X1R&amp;nbsp;</td><td>75004261</td><td></td></tr><tr><td>Densito 30PX densitometer</td><td>Mettler Toledo</td><td>MT51324450</td><td></td></tr><tr><td>ExoQuick solution&amp;nbsp;</td><td>Invitrogen</td><td>4484451</td><td></td></tr><tr><td>Polycarbonate thick-walled ultracentrifuge tube</td><td>Thermo Scientific</td><td>O3237</td><td></td></tr><tr><td>Sorvall WX100 ultracentrifuge&amp;nbsp;</td><td>Thermo Scientific</td><td>46902</td><td></td></tr><tr><td>Fat Red 7B&amp;nbsp;</td><td>Sigma-Aldrich</td><td>201618</td><td></td></tr><tr><td>&amp;beta;-mercaptoethanol&amp;nbsp;</td><td>Sigma-Aldrich</td><td></td><td></td></tr><tr><td>Amicon Ultra-15 Centrifugal filter devices 10K</td><td>Millipore</td><td>UFC901008</td><td></td></tr><tr><td>Amicon Ultra-centrifugal filter devices 3K</td><td>Millipore</td><td>UFC800308</td><td></td></tr><tr><td>QuickGel Lipo kit&amp;nbsp;</td><td>Helena Laboratories&amp;nbsp;</td><td>3344,3544T</td><td></td></tr><tr><td>Human lipoprotein standards for VLDL, LDL and HDL</td><td>LipoTrol; Helena Laboratories</td><td>5069</td><td></td></tr><tr><td>Rep Prep buffer&amp;nbsp;</td><td>Helena Laboratories&amp;nbsp;</td><td>3100</td><td></td></tr><tr><td>RNeasy MinElute spin columns&amp;nbsp;</td><td>Qiagen</td><td></td><td></td></tr><tr><td>NanoDrop 1000 analyzer</td><td>Thermo Scientific</td><td></td><td></td></tr><tr><td>miScript II RT Kit&amp;nbsp;</td><td>Qiagen</td><td>218161</td><td></td></tr><tr><td>CFX96 Touch real-time PCR detection system</td><td>BioRad</td><td></td><td></td></tr><tr><td>miRNeasy Serum/Plasma Kit</td><td>QIAGEN</td><td>217184</td><td></td></tr><tr><td>miScript Primer Assays</td><td>QIAGEN</td><td>141078139</td><td></td></tr><tr><td>miScript SYBR Green PCR Kit&amp;nbsp;</td><td>QIAGEN</td><td>218073</td><td></td></tr><tr><td>miRNeasy Serum/Plasma Spike-In Control</td><td>QIAGEN</td><td>219610</td><td></td></tr><tr><td>NaOH</td><td>SIGMA-ALDRICH</td><td>480878</td><td></td></tr><tr><td>0.20 &amp;#181;M sterile syringe filter</td><td>SIGMA-ALDRICH</td><td>Z227536</td><td></td></tr></tbody></table>

fast and simplified method for high through-put isolation of mirna from highly purified high density lipoprotein

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.

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</str...

Watch this Scientific Journal Video about Fast and Simplified Method for High Through-put Isolation of miRNA from Highly Purified High Density Lipoprotein at JoVE.com

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

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 ...

fast-simplified-method-for-high-through-put-isolation-mirna-from

Research

JoVE Journal

Biology

11.9K Views.  Virginia Commonwealth University.  MicroRNAs spelen een belangrijke regulerende rol en zijn in opkomst als nieuwe therapeutische doelen voor verschillende ziekten bij de mens. Het is aangetoond dat miRNAs worden vervoerd in high density lipoproteïnen. Wij hebben een eenvoudige methode om gezuiverd HDL geschikt voor miRNA analyse uit menselijk plasma snel isoleren ontwikkeld. 

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

Isolation of High-density Lipoproteins for Non-coding Small RNA Quantification

The diversity of small non-coding RNAs (sRNA) is rapidly expanding and their roles in biological processes, including gene regulation, are emerging. Most interestingly, sRNAs are also found outside of cells and are stably present in all biological fluids. As such, extracellular sRNAs represent a novel class of disease biomarkers and are likely involved in cell signaling and intercellular communication networks. To assess their potential as biomarkers, sRNAs can be quantified in plasma, urine, and other fluids. Nevertheless, to fully understand the impact of extracellular sRNAs as endocrine signals, it is important to determine which carriers are transporting and protecting them in biological fluids (e.g., plasma), which cells and tissues contribute to extracellular sRNA pools, and cells and tissues capable of accepting and utilizing extracellular sRNA. To accomplish these goals, it is critical to isolate highly pure populations of extracellular carriers for sRNA profiling and quantification. We have previously demonstrated that lipoproteins, particularly high-density lipoproteins (HDL), transport functional microRNAs (miRNA) between cells and HDL-miRNAs are significantly altered in disease. Here, we detail a new protocol that utilizes tandem HDL isolation with density-gradient ultracentrifugation (DGUC) and fast-protein-liquid chromatography (FPLC) to obtain highly pure HDL for downstream profiling and quantification of all sRNAs, including miRNAs, using both high-throughput sequencing and real-time PCR approaches. This protocol will be a valuable resource for the investigation of sRNAs on HDL.

This protocol describes the isolation and quantification of high-density lipoprotein small RNAs.

The diversity of small non-coding RNAs (sRNA) is rapidly expanding and their roles in biological processes, including gene regulation, are emerging. Most ...

Biochemistry

High Throughput MicroRNA Profiling: Optimized Multiplex qRT-PCR at Nanoliter Scale on the Fluidigm Dynamic ArrayTM IFCs

The broad involvement of miRNAs in critical processes underlying development, tissue homoeostasis and disease has led to a surging interest among the research and pharmaceutical communities. To study miRNAs, it is essential that the quantification of microRNA levels is accurate and robust. By comparing wild-type to small RNA deficient mouse embryonic stem cells (mESC), we revealed a lack of accuracy and robustness in previous published multiplex qRT-PCR techniques. Here, we describe an optimized method, including purifying away excessive primers from previous multiplex steps before singleplex real time detection, which dramatically increases the accuracy and robustness of the technique. Furthermore, we explain how performing the technique on a microfluidic chip at nanoliter volumes significantly reduces reagent costs and permits time effective high throughput miRNA expression profiling.

Here we describe an optimized multiplex reverse transcriptase quantitative PCR (qRT-PCR) protocol in combination with a microfluidic platform as a cost and time effective high-throughput screening tool for microRNA (miRNA) expression levels, especially when working with limited amounts of sample.

The broad involvement of miRNAs in critical processes underlying development, tissue homoeostasis and disease has led to a surging interest among the ...

Bioengineering

Enrichment of Native Lipoprotein Particles with microRNA and Subsequent Determination of Their Absolute/Relative microRNA Content and Their Cellular Transfer Rate

Lipoprotein particles are predominately transporters of lipids and cholesterol in the bloodstream. Furthermore, they contain small amounts of strands of noncoding microRNA (miRNA). In general, miRNA alters the protein expression profile due to interactions with messenger-RNA (mRNA). Thus, knowledge of the relative and absolute miRNA content of lipoprotein particles is essential to estimate the biological effect of cellular particle uptake. Here, a quantitative real-time polymerase chain reaction (qPCR)-based protocol is presented to determine the absolute miRNA content of lipoprotein particles&#8212;exemplified shown for native and miRNA-enriched lipoprotein particles. The relative miRNA content is quantified using multiwell microfluidic array cards. Furthermore, this protocol allows scientists to estimate the cellular miRNA and, thus, the lipoprotein particle uptake rate. A significant increase of the cellular miRNA level is observable when using high-density lipoprotein (HDL) particles artificially loaded with miRNA, whereas incubation with native HDL particles yields no significant effect due to their rather low miRNA content. In contrast, the cellular uptake of low-density lipoprotein (LDL) particles&#8212;neither with native miRNA nor artificially loaded with it&#8212;did not alter the cellular miRNA level.

Here, a quantitative real-time polymerase chain reaction-based protocol is presented for the determination of the native micro-RNA content (absolute/relative) of lipoprotein particles. In addition, a method for increasing the micro-RNA level, as well as a method for determining the cellular uptake rate of lipoprotein particles, is demonstrated.

Lipoprotein particles are predominately transporters of lipids and cholesterol in the bloodstream. Furthermore, they contain small amounts of strands of ...

Isolation and Analysis of Plasma Lipoproteins by Ultracentrifugation

Analysis of plasma lipoproteins and apolipoproteins is an essential part for the diagnosis of dyslipidemia and studies of lipid metabolism and atherosclerosis. Although there are several methods for analyzing plasma lipoproteins, ultracentrifugation is still one of the most popular and reliable methods. Because of its intact separation procedure, the lipoprotein fractions isolated by this method can be used for analysis of lipoproteins, apolipoproteins, proteomes, and functional study of lipoproteins with cultured cells in vitro. Here, we provide a detailed protocol to isolate seven lipoprotein fractions including VLDL (d&#60;1.006 g/mL), IDL (d=1.02 g/mL), LDLs (d=1.04 and 1.06 g/mL), HDLs (d=1.08, 1.10, and 1.21 g/mL) from rabbit plasma using sequential floating ultracentrifugation. In addition, we introduce the readers how to analyze apolipoproteins such as apoA-I, apoB, and apoE by SDS-PAGE and Western blotting and show representative results of lipoprotein and apolipoprotein profiles using hyperlipidemic rabbit models. This method can become a standard protocol for both clinicians and basic scientists to analyze lipoprotein functions.

Several methods have been used for analyzing plasma lipoproteins; however, ultracentrifugation is still one of the most popular and reliable methods. Here, we describe a method regarding how to isolate lipoproteins from plasma using sequential density ultracentrifugation and how to analyze the apolipoproteins for both diagnostic and research purposes.

Analysis of plasma lipoproteins and apolipoproteins is an essential part for the diagnosis of dyslipidemia and studies of lipid metabolism and ...

Isolation and Profiling of MicroRNA-containing Exosomes from Human Bile

Exosome research in the last three years has greatly extended the scope towards identification and characterization of biomarkers and their therapeutic uses.
Exosomes have recently been shown to contain microRNAs (miRs). MiRs themselves have arisen as valuable biomarkers for diagnostic purposes. As specimen collection in clinics and hospitals is quite variable, miRNA isolation from whole bile varies substantially. To achieve robust, accurate and reproducible miRNA profiles from collected bile samples in a simple manner required the development of a high-quality protocol to isolate and characterize exosomes from bile. The method requires several centrifugations and a filtration step with a final ultracentrifugation step to pellet the isolated exosomes. Electron microscopy, Western blots, flow cytometry and multi-parameter nanoparticle optical analysis, where available, are crucial characterization steps to validate the quality of the exosomes. For the isolation of miRNA from these exosomes, spiking the lysate with a non-specific, synthetic miRNA from a species like Caenorhabditis elegans, i.e., Cel-miR-39, is important for normalization of RNA extraction efficiency. The isolation of exosome from bile fluid following this method allows the successful miRNA profiling from bile samples stored for several years at -80 &#176;C.

Bile fluid is a valuable source of extracellular vesicles/exosomes that contain potentially important biomarkers. This protocol represents a robust method to isolate exosomes from human bile for further analyses including miRNA profiling.

Exosome research in the last three years has greatly extended the scope towards identification and characterization of biomarkers and their therapeutic ...

Medicine

Purification and microRNA Profiling of Exosomes Derived from Blood and Culture Media

Stable miRNAs are present in all body fluids and some circulating miRNAs are protected from degradation by sequestration in small vesicles called exosomes. Exosomes can fuse with the plasma membrane resulting in the transfer of RNA and proteins to the target cell. Their biological functions include immune response, antigen presentation, and intracellular communication. Delivery of miRNAs that can regulate gene expression in the recipient cells via blood has opened novel avenues for target intervention. In addition to offering a strategy for delivery of drugs or RNA therapeutic agents, exosomal contents can serve as biomarkers that can aid in diagnosis, determining treatment options and prognosis.	Here we will describe the procedure for quantitatively analyzing miRNAs and messenger RNAs (mRNA) from exosomes secreted in blood and cell culture media. Purified exosomes will be characterized using western blot analysis for exosomal markers and PCR for mRNAs of interest. Transmission electron microscopy (TEM) and immunogold labeling will be used to validate exosomal morphology and integrity. Total RNA will be purified from these exosomes to ensure that we can study both mRNA and miRNA from the same sample. After validating RNA integrity by Bioanalyzer, we will perform a medium throughput quantitative real time PCR (qPCR) to identify the exosomal miRNA using Taqman Low Density Array (TLDA) cards and gene expression studies for transcripts of interest.	
 These protocols can be used to quantify changes in exosomal miRNAs in patients, rodent models and cell culture media before and after pharmacological intervention. Exosomal contents vary due to the source of origin and the physiological conditions of cells that secrete exosomes. These variations can provide insight on how cells and systems cope with stress or physiological perturbations. Our representative data show variations in miRNAs present in exosomes purified from mouse blood, human blood and human cell culture media.	
 Here we will describe the procedure for quantitatively analyzing miRNAs and messenger RNAs (mRNA) from exosomes secreted in blood and cell culture media. Purified exosomes will be characterized using western blot analysis for exosomal markers and PCR for mRNAs of interest. Transmission electron microscopy (TEM) and immunogold labeling will be used to validate exosomal morphology and integrity. Total RNA will be purified from these exosomes to ensure that we can study both mRNA and miRNA from the same sample. After validating RNA integrity by Bioanalyzer, we will perform a medium throughput quantitative real time PCR (qPCR) to identify the exosomal miRNA using Taqman Low Density Array (TLDA) cards and gene expression studies for transcripts of interest.
 These protocols can be used to quantify changes in exosomal miRNAs in patients, rodent models and cell culture media before and after pharmacological intervention. Exosomal contents vary due to the source of origin and the physiological conditions of cells that secrete exosomes. These variations can provide insight on how cells and systems cope with stress or physiological perturbations. Our representative data show variations in miRNAs present in exosomes purified from mouse blood, human blood and human cell culture media

The presence of stable microRNAs (miRNAs) in exosomes has generated immense interest as a novel mode of intercellular communication, for their potential utility as biomarkers and as a route for therapeutic intervention. Here we demonstrate exosome purification from blood and culture media followed by quantitative PCR to identify miRNAs being transported.

Stable miRNAs are present in all body fluids and some circulating miRNAs  are protected from degradation by sequestration in small vesicles called  ...

Digital PCR for Quantifying Circulating MicroRNAs in Acute Myocardial Infarction and Cardiovascular Disease

Circulating serum microRNAs (miRNAs) have shown promise as biomarkers for the cardiovascular disease and acute myocardial infarction (AMI), being released from the cardiovascular cells into the circulation. Circulating miRNAs are highly stable and can be quantified. The quantitative expression of specific miRNAs can be linked to the pathology, and some miRNAs show high tissue and disease specificity. Finding novel biomarkers for cardiovascular diseases is of importance for medical research. Quite recently, digital polymerase chain reaction (dPCR) has been invented. dPCR, combined with fluorescent hydrolysis probes, enables specific direct absolute quantification. dPCR exhibits superior technical qualities, including a low variability, high linearity, and high sensitivity compared to the quantitative polymerase chain reaction (qPCR). Thus, dPCR is a more accurate and reproducible method for directly quantifying miRNAs, particularly for the use in large multi-center cardiovascular clinical trials. In this publication, we describe how to effectively perform digital PCR in order to assess the absolute copy number in serum samples.

Circulating microRNAs have shown promise as biomarkers for cardiovascular diseases and acute myocardial infarctions. In this study, we describe a protocol for miRNA extraction, reverse transcription, and digital PCR for the absolute quantification of miRNAs in the serum of patients with cardiovascular disease.

Circulating serum microRNAs (miRNAs) have shown promise as biomarkers for the cardiovascular disease and acute myocardial infarction (AMI), being released ...

Isolation of Small Noncoding RNAs from Human Serum

The analysis of RNA and its expression is a common feature in many laboratories. Of significance is the emergence of small RNAs like microRNAs, which are found in mammalian cells. These small RNAs are potent gene regulators controlling vital pathways such as growth, development and death and much interest has been directed at their expression in bodily fluids. This is due to their dysregulation in human diseases such as cancer and their potential application as serum biomarkers. However, the analysis of miRNA expression in serum may be problematic. In most cases the amount of serum is limiting and serum contains low amounts of total RNA, of which small RNAs only constitute 0.4-0.5%1. Thus the isolation of sufficient amounts of quality RNA from serum is a major challenge to researchers today. In this technical paper, we demonstrate a method which uses only 400 &#181;l of human serum to obtain sufficient RNA for either DNA arrays or qPCR analysis. The advantages of this method are its simplicity and ability to yield high quality RNA. It requires no specialized columns for purification of small RNAs and utilizes general reagents and hardware found in common laboratories. Our method utilizes a Phase Lock Gel to eliminate phenol contamination while at the same time yielding high quality RNA. We also introduce an additional step to further remove all contaminants during the isolation step. This protocol is very effective in isolating yields of total RNA of up to 100 ng/&#181;l from serum but can also be adapted for other biological tissues.

This protocol describes a method for extracting small RNAs from human serum. We have used this method to isolate microRNAs from cancer serum for use in DNA arrays and also singleplex quantitative PCR. The protocol utilizes phenol and guanidinium thiocyanate reagents with modifications to yield high quality RNA.

The analysis of RNA and its expression is a common feature in many laboratories. Of significance is the emergence of small RNAs like microRNAs, which are ...

Highly Efficient Ligation of Small RNA Molecules for MicroRNA Quantitation by High-Throughput Sequencing

MiRNA cloning and high-throughput sequencing, termed miR-Seq, stands alone as a transcriptome-wide approach to quantify miRNAs with single nucleotide resolution. This technique captures miRNAs by attaching 3&#8217; and 5&#8217; oligonucleotide adapters to miRNA molecules and allows de novo miRNA discovery. Coupling with powerful next-generation sequencing platforms, miR-Seq has been instrumental in the study of miRNA biology. However, significant biases introduced by oligonucleotide ligation steps have prevented miR-Seq from being employed as an accurate quantitation tool. Previous studies demonstrate that biases in current miR-Seq methods often lead to inaccurate miRNA quantification with errors up to 1,000-fold for some miRNAs1,2. To resolve these biases imparted by RNA ligation, we have developed a small RNA ligation method that results in ligation efficiencies of over 95% for both 3&#8217; and 5&#8242; ligation steps. Benchmarking this improved library construction method using equimolar or differentially mixed synthetic miRNAs, consistently yields reads numbers with less than two-fold deviation from the expected value. Furthermore, this high-efficiency miR-Seq method permits accurate genome-wide miRNA profiling from in vivo total RNA samples2.

MicroRNAs (miRNAs) are a widely conserved class of regulatory molecules. Here we describe a miRNA cloning method that relies upon two potent ligation steps followed by high-throughput sequencing. Our method permits accurate genome-wide quantitation of miRNAs.

MiRNA cloning and high-throughput sequencing, termed miR-Seq, stands alone as a transcriptome-wide approach to quantify miRNAs with single nucleotide ...

Circulating miRNA Extraction: A Method to Isolate miRNA from Plasma Samples Via Organic Extraction and Small RNA Enrichment

Source: Canale, M. et al., <a href="https://www.jove.com/v/58615"> Detection of a Circulating MicroRNA Custom Panel in Patients with Metastatic Colorectal Cancer.</a> J. Vis. Exp. (2019).
This video describes the extraction of circulating microRNA from plasma samples of colorectal cancer patients using organic extraction followed by glass-fiber column enrichment. miRNAs are stable in biological fluids such as serum and plasma, which renders them ideal circulating biomarkers for cancer diagnosis, prognosis, and treatment decision-making and monitoring. This method will help identify new circulating biomarkers capable of predicting clinical outcomes in patients.

Source: Canale, M. et al.,  Detection of a Circulating MicroRNA Custom Panel in Patients with Metastatic Colorectal Cancer. J. Vis. Exp. (2019).
This ...

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

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