Name: fast and simplified method for high through-put isolation of mirna from highly purified high density lipoprotein
Uploaded: 2016-07-27T13:00:00
Description: 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

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. Raccolta dei campioni di sangue <ol><li> Raccogliere digiuno periferico campioni di sangue venoso in apposite provette 10 ml di plastica contenenti acido anticoagulante etilendiamminotetraacetico (EDTA) (che ha diversi vantaggi rispetto ad altri anticoagulanti) per venopuntura standard di una vena di primo piano nella fossa antecubitale. </li><li> Centrifugare i campioni di sangue a 1.600 xg per 20 min a 4 ° C in una centrifuga da tavolo per ottenere plasma privo di globuli rossi e piccole quantità di RNA. </li><...

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L&#39;identificazione di nuovi biomarcatori dal sangue sarà di aiuto nella diagnosi clinica e la prognosi di varie malattie. I microRNA sono noti per possedere tutte le qualità di biomarcatori e hanno dimostrato in vari studi 14-17. In questo studio abbiamo dimostrato metodo facile rapida e semplice per isolare miRNA da HDL plasma. Densità convenzionale gradiente metodo ultra-centrifugazione di isolamento di VLDL, LDL e HDL dipende accurata campionamento di plasma, preparazione precisa della soluzione tamp...

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.

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		<tr height="21">
			<td height="21" style="height:21px;width:348px;">Plastic Vacutainer Lavender K2EDTA tubes&#160;</td>
			<td style="width:279px;">Becton, Dickinson and Company</td>
			<td style="width:172px;">366643</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Centrifuge</td>
			<td>Thermo Scientific, Sorvall Legend X1R&#160;</td>
			<td style="width:172px;">75004261</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Densito 30PX densitometer</td>
			<td>Mettler Toledo</td>
			<td style="width:172px;">MT51324450</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ExoQuick solution&#160;</td>
			<td>Invitrogen</td>
			<td style="width:172px;">4484451</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Polycarbonate thick-walled ultracentrifuge tube</td>
			<td>Thermo Scientific</td>
			<td style="width:172px;">O3237</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sorvall WX100 ultracentrifuge&#160;</td>
			<td>Thermo Scientific</td>
			<td style="width:172px;">46902</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fat Red 7B&#160;</td>
			<td>Sigma-Aldrich</td>
			<td style="width:172px;">201618</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#946;-mercaptoethanol&#160;</td>
			<td>Sigma-Aldrich</td>
			<td style="width:172px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Amicon Ultra-15 Centrifugal filter devices 10K</td>
			<td>Millipore</td>
			<td style="width:172px;">UFC901008</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Amicon Ultra-centrifugal filter devices 3K</td>
			<td>Millipore</td>
			<td style="width:172px;">UFC800308</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">QuickGel Lipo kit&#160;</td>
			<td>Helena Laboratories&#160;</td>
			<td style="width:172px;">3344,3544T</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Human lipoprotein standards for VLDL, LDL and HDL</td>
			<td>LipoTrol; Helena Laboratories</td>
			<td style="width:172px;">5069</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Rep Prep buffer&#160;</td>
			<td>Helena Laboratories&#160;</td>
			<td style="width:172px;">3100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RNeasy MinElute spin columns&#160;</td>
			<td>Qiagen</td>
			<td style="width:172px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NanoDrop 1000 analyzer</td>
			<td>Thermo Scientific</td>
			<td style="width:172px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">miScript II RT Kit&#160;</td>
			<td>Qiagen</td>
			<td style="width:172px;">218161</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CFX96 Touch real-time PCR detection system</td>
			<td>BioRad</td>
			<td style="width:172px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:348px;">miRNeasy Serum/Plasma Kit</td>
			<td style="width:279px;">QIAGEN</td>
			<td style="width:172px;">217184</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:348px;">miScript Primer Assays</td>
			<td style="width:279px;">QIAGEN</td>
			<td style="width:172px;">141078139</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:348px;">miScript SYBR Green PCR Kit&#160;</td>
			<td style="width:279px;">QIAGEN</td>
			<td style="width:172px;">218073</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:348px;">miRNeasy Serum/Plasma Spike-In Control</td>
			<td style="width:279px;">QIAGEN</td>
			<td style="width:172px;">219610</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.

Isolamento di lipoproteine ​​ad alta densità dopo la rimozione di Esosomi Per ottenere miRNA da HDL altamente purificato è necessario rimuovere esosomi che rappresentano una fonte di contaminazione miRNA 7. Ciò è stato fatto prima gradiente di densità ultracentrifugazione con un kit disponibile in commercio. Per scopi pratici standard gradiente di densità protocollo ultracentrifugazione tre fasi sviluppato dalla società comme...

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

I microRNA svolgono un ruolo regolatore importante e stanno emergendo come nuovi bersagli terapeutici per varie malattie umane. E &#39;stato dimostrato che i miRNA sono effettuate in lipoproteine ​​ad alta densità. Abbiamo sviluppato un metodo semplificato per isolare rapidamente HDL purificata per l&#39;analisi miRNA dal plasma umano.

Veloce e semplificato Metodo per alta through-put Isolamento dei miRNA da altamente purificato lipoproteine ​​ad alta densità

Small non-coding RNAs (miRNAs) have been implicated in a variety of human diseases including metabolic syndromes. They may be utilized as biomarkers for ...

The overall goal of this method is to develop a simplified approach to rapidly isolate purified high density lipoproteins from human blood plasma for micro RNA analysis. This method can help answer key questions in hepatology and cardiology, such as understanding the molecular mechanisms and protective functions of high density lipoproteins. Some main advantages of this technique is that purified HDL micro RNA come pre-isolated from less volume of samples within a short period of time.In this protocol, plasma is obtained from fasting peripheral venous blood samples by multiple centrifugation steps as described in the protocol text. Transfer the plasma to a new tube. And measure the density of the plasma using a densitometer at room temperature, per the manufacturer's instructions.To remove the circulating exosomes that represent a source of micro RNA contamination, add 252 microliters of exosome precipitation solution to one milliliter of plasma. And incubate for 30 minutes at four degrees Celsius. Then centrifuge the mixture for 30 minutes to pellet out the exosomes.Transfer one milliliter of the resulting supernatant to a polycarbonate thick walled ultracentrifuge tube for further processing, as demonstrated in the next video segment. Isolation of high density lipoproteins, or HDL, is accomplished by a three step density gradient ultracentrifugation protocol, that involves a sequential isolation of very low density lipoproteins, or VLDL, low density lipoproteins, or LDL, and HDL. The three different density solutions, A, B, and C, must be prepared fresh, following the procedure described in the protocol text.In a 6.5 milliliter polycarbonate thick walled ultracentrifuge tube, mix one milliliter of plasma and 200 microliters of nuclease-free Fat Red 7B. Then, carefully layer five milliliters 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.Load the tubes into a pre-chilled rotor. Place the rotor into a floor centrifuge and centrifuge for two hours. At the end of the run, two layers should be visible.Remove 1.5 milliliters of the top layer, representing the VLDL fraction. And store it four degrees Celsius. Use a pipette to transfer four milliliters from the bottom of the tube to a new 50 milliliter tube.This contains the LDL and HDL fraction. The next step is the isolation of LDL. Mix two milliliters of solution B and 100 microliters of nuclease-free Fat Red 7B into the tube containing the LDL and HDL fraction.Put on ice for five to ten minutes. Transfer 6.5 milliliters of the mixed sample into a polycarbonate thick walled ultracentrifuge tube. Load the tube into a pre-chilled rotor and centrifuge for three hours.When the centrifugation is complete, remove 1.5 milliliters of the top layer, representing the LDL fraction and keep at four degrees Celsius. Transfer four milliliters from the bottom of the tube, containing the HDL fraction to a 50 milliliter sterile tube. The third step is the isolation of HDL.Mix two milliliters of solution C, 100 microliters of nuclease-free Fat Red 7B, and 15 microliters of 98%beta mercaptoethanol into the tube, containing the HDL fraction. Check the density of the mixture. Load the tube into a pre-chilled rotor and centrifuge for three hours.After that, remove two milliliters of the top layer, representing the HDL fraction, and keep at four degrees Celsius. Excessive salt must be removed from the lipoprotein fractions to avoid interference with subsequent agarose gel electrophoresis and PCR. In this video, desalting and concentration will be demonstrated for only the HDL fraction.Add 13 milliliters of cold PBS to the HDL fraction and transfer to a centrifugal filter device with the molecular weight cut-off of 10K. Centrifuge in a swinging bucket rotor, at 4000 times g, and four degrees Celsius, for 30 minutes. Desalt the HDL fraction, a second time, using 13 milliliters of cold PBS, and centrifuging as before.After centrifugation, remove the lipoprotein containing solutes, and keep at four degrees Celsius. To assess the quality and purity of the isolated lipoproteins, agarose gel electrophoresis was performed with human lipoprotein standards, included as a size reference. Representative results from an isolation, without beta mercaptoethanol, showed that the HDL fraction is devoid of any contamination of VLDL and exhibits typical alpha mobility.However, the HDL fraction also shows traces of beta mobility due to contamination with lipoproteins. The addition of beta mercaptoethanol to solution C, during the last centrifugation step, effectively removes all contaminating lipoproteins from the HDL fraction. To demonstrate the feasibility of quantifying micro RNAs, carried in human HDL, purified with this method, two different micro RNAs were amplified by real-time quantitative PCR.In these amplification plots, the horizontal lines represent the detection threshold. Representative real-time PCR data from isolated HDL, obtained from six samples, showed the consistent detection of both micro RNAs. Lastly, melting curve analyses clearly show a distinct single peak for both micro RNAs, consistent with specific amplification in the preceding PCR.Once mastered, this technique can be done in nine hours, together with the desalting procedures, if it is done properly. While attempting this procedure, it is important to work at four to eight degrees centigrade, at all times. And to adjust the density of each lipoprotein fraction before ultracentrifugation.Following this procedure, other methods, such as electron microscopy, can be performed in order to answer additional questions, like the purity of HDL. After its development, this technique will allow researchers, in the field of metabolic syndrome, to explore HDL-associated micro RNAs as bio markers, in humans with suspected fatty liver disease. It may also be used to better understand the function of HDL and the molecular mechanisms by which it alters cell biology.

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