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>

<ol>
	<li>Bartel, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNAs:+genomics,+biogenesis,+mechanism,+and+function.">MicroRNAs: genomics, biogenesis, mechanism, and function.</a> Cell. 116 (2), 281-297 (2004).</li><li>Arroyo, J. D., 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=Argonaute2+complexes+carry+a+population+of+circulating+microRNAs+independent+of+vesicles+in+human+plasma.">Argonaute2 complexes carry a population of circulating microRNAs independent of vesicles in human plasma.</a> Proc Natl Acad Sci U S A. 108 (12), 5003-5008 (2011).</li><li>Vickers, K. 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. J., Moore, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNA+control+of+high-density+lipoprotein+metabolism+and+function.">MicroRNA control of high-density lipoprotein metabolism and function.</a> Circ Res. 114 (1), 183-192 (2014).</li><li>Raposo, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exosomes:+endosomal-derived+vesicles+shipping+extracellular+messages.">Exosomes: endosomal-derived vesicles shipping extracellular messages.</a> Curr Opin Cell Biol. 16 (4), 415-421 (2004).</li><li>Redgrave, T. G., Roberts, D. C., West, C. 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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 density and quantitative transfer of bottom lipoprotein fractions8. There are numerous methods that have been described to isolate HDL8 -11. These methods are often either very laborious or require large volume of plasma and extensive dialysis for desalting isolated lipoproteins and do not completely remove exosomes and lipoproteins as a source of miRNAs 3. To address these shortcomings we have established this simple and fast method of isolation of miRNA from plasma HDL.
The key goal and primary purpose of this study was to separate and isolate HDL-miRNA complex by ultracentrifugation without RBCs, buffy coat, exosomes, secretory vesicles, VLDL, LDL and Lipoproteins (a) interference, then isolate miRNAs from purified HDL. Contamination and interference of any of these components with HDL will alter the expected experimental results and miRNA profiling data. Isolation of DNA, RNA and miRNAs from blood is a very difficult task due to its complex nature. It has always been posed with lot of technical challenges to nucleic acids (including miRNAs) extraction and purification compared to cells, tissues and organ samples18. Also there are relatively less miRNA genes present in circulating blood cells. Blood is a known carrier for exosomes, secretory vesicles and HDLs along with free circulating miRNAs, which are relatively less18. Therefore, larger amount of blood plasma sample volume is required for processing and isolation of HDL-miRNA complex. In addition, the short nature of the miRNAs and their target sequences, makes it even difficult to achieve sufficient specificity with standard PCR oligonucleotide technologies. The cellular components of blood are always know to secrete miRNA and mRNA in response to change in external environment including temperature, microorganisms and toxins or stress. Also presence of nucleases, RNases, circulating proteins and other enzymes in blood will affect these miRNA isolation.
The circulating miRNA amplification also lacks a well-established known housekeeping genes like &#946;-actin or GAPDH for data normalization while using the &#916;&#916;Ct method of qRT-PCR. This again poses one of the major hurdle for miRNA profiling from circulating blood serum or plasma19. Commonly used short non coding snoRNAs and snRNAs are rarely expressed in blood and this makes it further difficult in utilizing them as internal control genes. To solve these demerits of snoRNAs and snRNAs and to avoid any technical difficulties we used Serum/Plasma synthetic Spike-In control in the assays as internal control gene as followed according to the manufacturer protocol. It helps in normalization for any nonspecific amplification and changes occurred during miRNA purification and PCR reaction18. Even in the case of degradation of miRNA or absence of any specific miRNA amplification signals, the spike-in control should amplify in qRT-PCR reactions and give an expected signal with a reasonably constant and uniform Ct value. Based on the spike in control amplification along with three negative controls and positive controls we got good optimal data validation for miR-223 gene. This confirmed that we have got good yield of miR-223 from circulating plasma HDL from this simple method.
In conclusion, we have established a method of density gradient ultracentrifugation (DGUC) with the addition of &#946;-mercaptoethanol for purification of miRNA from plasma HDL. The method has several advantages: (a) VLDL, LDL and HDL are separated within a short period of time by floor ultracentrifuge compare to other methods that needs 24 - 96 hr 8-11; (b) LDL and HDL dialysis, desalting/concentrating, take place within 1 hr compare with other methods that took 24 or more hours8,9; (c) Contamination of Lipoproteins is removed by &#946;-mercaptoethanol in HDL fraction, but is not intended to be used in the LDL fraction; (d) The sample volume required is only 1 ml for all stepwise VLDL, LDL and HDL isolations compare with the other methods that needs more specimen volume; (e) It minimized oxidative damage to HDL during isolation11; (f) A maximum of 12 samples can be analyzed in a single lipoprotein separation analytical run; (g) The 250 &#181;l sample volume of extracted HDL is enough to analyse miRNA concentration/purity, agarose gel electrophoresis, protein concentration, microarray and RT-qPCR procedures. The limitations of our method is that small percentage of HDL-miRNA may lose during the exosome precipitation step and the required plasma sample volume should be more than 200 &#181;l to isolate HDL-miRNA.
Finally, the plasma microRNAs are not only derived from damaged or renegade blood cells in the circulation but also from healthy normal blood cells and cells from other parts of the body which includes various healthy and diseased tissues and organs affected by ongoing health status of the body 20. This present study has systematically purified mature miR-223 from isolated HDL of human plasma. This study clearly demonstrates that levels of mature miR-223 in the highly purified HDL plasma are detectable, stable, reproducible and consistent among individuals sample, thereby greatly facilitating clinical use of futurity tests for lipoproteins, liver, cardiovascular and other metabolic syndromes. Surprisingly, mature miRNAs, particularly miR-223 from HDL plasma are probably resistant to nuclease digestion and other harsh conditions which potentially explain the stability of miR-223 in purified HDL. The mechanism of resistance of miR-223 to RNases requires further study. Obviously, studying miRNA expression profiles in these purified HDL plasma would shed light on future miRNA markers in studying various maladies. These HDL associated microRNAs in the plasma can be used as a potential clinical diagnostic biomarkers and personalized medicine in the future.

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 border="0" cellpadding="0" cellspacing="0" width="965">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<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.

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). This protocol requires centrifugation with a fixed-angle rotor at a speed of 140,000 rpm which is substantially faster than commonly used protocols with centrifugation forces up to 54,000 rpm3, 8, 9, and 10. Employing a widely available T-1270 rotor with a maximum force of 70,000 rpm, centrifugation was initially carried out with polyallomer tubes which have failed to resist the centrifugation forces. To avoid collapse of tubes, polycarbonate tubes were successfully used. Centrifugation time is critical for lipoprotein oxidation and potentially miRNA degradation11. Therefore several different centrifugation times, ranging from a total of 8 to 96 hr were tested. Furthermore temperature at which centrifugation was carried out was adjusted based on centrifugation time and force, respectively.
Purity of the High Density Lipoprotein Fractions 
Purity of isolated high density lipoprotein fractions were checked with agarose gel electrophoresis. Figure 2. illustrates a typical electrophoresis result for the HDL subtraction isolated by gradient ultracentrifugation. It clearly showed that HDL was devoid of any contamination of VLDL and exhibits typical &#945;-mobility. The absence of any &#945;-migrating lipoproteins in the VLDL and LDL fractions demonstrates the complete recovery from HDL during the ultracentrifugation step. The HDL fraction, however, also showed some trace of &#946;-mobility, a known electrophoretic banding pattern due to contamination with lipoproteins (Lp(a))12.
Elimination of Lp(a) from Isolated HDL 
Studying miRNAs carried in HDL requires isolation of HDL of highest purity. Interference of Lp(a) with HDL potentially contributes to cross-contamination of HDL with miRNAs carried in LDL. To minimize this contamination of HDL, 15 &#956;l of &#946;-mercaptoethanol 10&#160; was added to solution C during the last centrifugation step (Figure 1). Addition of &#946;-mercaptoethanol has been shown not to change HDL density properties10. As displayed in Figure 3., addition of &#946;-mercaptoethanol resulted in the absence of any b-migrating lipoproteins consistent with very effective removal of Lp (a).
Purification of miRNA from HDL 
Isolation of miRNA was initially attempted employing lysis reagent which is commonly used for RNA extraction from blood. Although this method resulted in a very good RNA yield (91.45 ng/&#181;l) but after spectrophotometric analysis showed unsatisfactory RNA purity. Additional purification of the isolated RNA by removing phenol improved the purity but the RNA yield was now significantly low. Next, another kit was tested which showed acceptable RNA purity (260/280 nm ratio of 1.7) but the RNA yield was 26-fold lower compared with the lysis reagent (3.45 vs. 91.45 ng/&#181;l). The best results for acceptable RNA purity with a good yield were obtained with the miRNeasy serum/plasma kit (48.2 ng/&#181;l; 260/280 nm ratio of 1.6) and therefore this extraction procedure was subsequently employed for the detection of miRNA from the isolated HDL lipoprotein fraction.
To demonstrate the feasibility to quantify miRNAs carried in human HDL purified with this method, we chose to amplify miR-223. This miRNA was chosen because miR-223 was identified in cargo of HDL3 and was shown to repress HDL cholesterol uptake5. The Figure 4A. shows a typical log plot of amplification curves comparing baseline threshold and threshold cycle values after optimization of the PCR reaction for the miR-223 and the spiked-in Cel-mir-39. This synthetic gene was used as internal control as there are no reports of known normalization control for miRNA in plasma. In addition, several potential established endogenous housekeeping genes like RNU6-2, RNU-48, HY3 could not be detected and SNORD95 could be detected in purified HDL, but, the difference in Ct values of miR-223 and SNORD95 is less than five (data not shown). Melting curve analysis illustrated in Figure 5. clearly shows a distinct single peak consistent with amplification of a selective miRNA in the preceding PCR. As illustrated in Figure 4B., both miR-223 and the reference Cel-mir-39 could consistently be detected in all six pro bands. Relatively small variations among all individuals were observed for Cel-mir-39 compared with miR-223. These findings support isolation of HDL at high purity to allow detection of its miRNA cargo.
Detection of miR-223 in Purified HDL 
Real time quantitative PCR method was used to quantify double hairpin-structure miRNA precursors to single strand miRNA. This method needs a forward/reverse gene-specific primers and a thermostable reverse transcriptase to convert the hairpin structure of the miRNA to cDNA. The cDNA was subsequently amplified and quantified using real-time qPCR with the help of SYBR green detection. MicroRNA from serum, plasma and purified HDL plasma can be accurately profiled using the miRNA RT PCR system.
Our experimental product of mean miR-223 gene expression showed a Ct value of 30.9 in purified HDL and its corresponding negative controls were above cut-off 35 (NTC = 38.1 Ct value, NRT and NAC were not amplified). In this experiment, we saw in the purified HDL samples the formation of Primer-dimer with low melting temperature (73 &#176;C in miR-223 and 75.5 &#176;C in Cel-miR-39) and higher Ct value than the specific miRNA assays (Table 1). The Primer-dimers in Table 1. occur probably because of high concentration of primers in the solution. This occurs mostly when primer molecules attach to each other after 30 PCR cycles 13. This allows them to anneal to other primer molecules and in effect, become linear mini-templates and it will cause higher background and may lead to a generation of Ct value &#60; 40 for NTC (No template control) samples.
<img alt="Figure 1" src="/files/ftp_upload/54257/54257fig1.jpg" /> 
Figure 1. Schematic Representation of the HDL Isolation Procedure. HDL was prepared by density gradient ultracentrifugation in a series of three centrifugation steps. Distribution of lipoprotein bands and intermediate fractions in the density gradient are illustrated. <a href="https://www.jove.com/files/ftp_upload/54257/54257fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/54257/54257fig2.jpg" /> 
Figure 2. Agarose Gel Electrophoresis of Lipoproteins Isolated Without &#946;-mercaptoethanol. VLDL, LDL and HDL fractions isolated by density gradient ultracentrifugation without adding &#946;-mercaptoethanol to solution C demonstrate the presence of Lp (a) in the HDL fraction. Lanes 1, 2, 9, and 10 each represent the size marker; Lanes 3/4, 5/6 and 7/8 represent VLDL, LDL and HDL respectively. <a href="https://www.jove.com/files/ftp_upload/54257/54257fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/54257/54257fig3.jpg" /> 
Figure 3. Agarose Gel Electrophoresis of HDL Isolated with &#946;-mercaptoethanol. Adding &#946;-mercaptoethanol to solution C before the last centrifugation step resulted in the removal of Lp(a) from the HDL fraction. Lanes 1, 2, 9 and 10 each represent the size marker; Lanes 3-8 represent HDL. <a href="https://www.jove.com/files/ftp_upload/54257/54257fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/54257/54257fig4.jpg" /> 
Figure 4. Amplification Plot of Two Different miRNAs and Expression of Cel-miR-39 and miR-223. (A) Real-time quantitative PCR employing isolated HDL was carried out as described. The PCR was run for 45 cycles and the point at which the curve intersects the threshold is the CT-Value. The data show the expression of Cel-miR-39 (left panel) and miR-223 (right panel), respectively. The horizontal line represents the detection threshold. A cycle number of 35 was set as cutoff for positive amplification. (B) Representative real-time PCR data from isolated HDL obtained from 6 samples are shown. Raw mean Ct values are shown for Cel-miR-39 and miR-223, whose expression levels vary less than 2 Ct value in purified HDL plasma fraction between 6 samples, each from a different donor. Expression profiling was performed using the PCR System and the Human Serum &#38; Plasma miRNA qRT-PCR. <a href="https://www.jove.com/files/ftp_upload/54257/54257fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/54257/54257fig5.jpg" /> 
Figure 5. Melting Curves for Cel-miR-39 and miR-223. As illustrated, the presence of a single peak indicates specific amplification of Cel-miR-39 (right panel) and miR-223 (left panel), respectively. <a href="https://www.jove.com/files/ftp_upload/54257/54257fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Table 1" src="/files/ftp_upload/54257/54257table1.jpg" /> 
Table 1. Row Ct value of negative and positive control of synthetic Cel-miR-39 and miR-223 characterized in serum, plasma, cDNA, and purified HDL fraction. NTC (No template control), NRT (No reverse transcription enzyme), NAC (No amplification control, only water and reagents of qRT-PCR).

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

Nanyang Technological University

Research

JoVE Journal

Biology

11.8K Views.  Virginia Commonwealth University. 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.

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

A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing measurement of multiple cellular phenotypes within a single assay. In this study, we utilized HCA to develop a novel assay for neurotoxicity. Neurotoxicity assessment represents an important part of drug safety evaluation, as well as being a significant focus of environmental protection efforts. Additionally, neurotoxicity is also a well-accepted in vitro marker of the development of neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Recently, the application of HCA to neuronal screening has been reported. By labeling neuronal cells with &beta;III-tubulin, HCA assays can provide high-throughput, non-subjective, quantitative measurements of parameters such as neuronal number, neurite count and neurite length, all of which can indicate neurotoxic effects. However, the role of astrocytes remains unexplored in these models. Astrocytes have an integral role in the maintenance of central nervous system (CNS) homeostasis, and are associated with both neuroprotection and neurodegradation when they are activated in response to toxic substances or disease states. GFAP is an intermediate filament protein expressed predominantly in the astrocytes of the CNS. Astrocytic activation (gliosis) leads to the upregulation of GFAP, commonly accompanied by astrocyte proliferation and hypertrophy. This process of reactive gliosis has been proposed as an early marker of damage to the nervous system. The traditional method for GFAP quantitation is by immunoassay. This approach is limited by an inability to provide information on cellular localization, morphology and cell number. We determined that HCA could be used to overcome these limitations and to simultaneously measure multiple features associated with gliosis - changes in GFAP expression, astrocyte hypertrophy, and astrocyte proliferation - within a single assay. In co-culture studies, astrocytes have been shown to protect neurons against several types of toxic insult and to critically influence neuronal survival. Recent studies have suggested that the use of astrocytes in an in vitro neurotoxicity test system may prove more relevant to human CNS structure and function than neuronal cells alone. Accordingly, we have developed an HCA assay for co-culture of neurons and astrocytes, comprised of protocols and validated, target-specific detection reagents for profiling &beta;III-tubulin and glial fibrillary acidic protein (GFAP). This assay enables simultaneous analysis of neurotoxicity, neurite outgrowth, gliosis, neuronal and astrocytic morphology and neuronal and astrocytic development in a wide variety of cellular models, representing a novel, non-subjective, high-throughput assay for neurotoxicity assessment. The assay holds great potential for enhanced detection of neurotoxicity and improved productivity in neuroscience research and drug discovery.

This article describes a novel protocol and reagent set designed for sensitive measurement of neurotoxic effects of compounds and treatments on co-cultures of neurons and astrocytes using high content analysis. Results demonstrate that high content analysis represents an exciting novel technology for neurotoxicity assessment.

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing ...

Quantification of dsDNA using the Hitachi F-7000 Fluorescence Spectrophotometer and PicoGreen Dye

Quantification of DNA, especially in small concentrations, is an important task with a wide range of biological applications including standard molecular biology assays such as synthesis and purification of DNA, diagnostic applications such as quantification of DNA amplification products, and detection of DNA molecules in drug preparations. During this video we will demonstrate the capability of the Hitachi F-7000 Fluorescence Spectrophotometer equipped with a Micro Plate Reader accessory to perform dsDNA quantification using Molecular Probes Quant-it PicoGreen dye reagent kit.
The F-7000 Fluorescence Spectrophotometer offers high sensitivity and high speed measurements. It is a highly flexible system capable of measuring fluorescence, luminescence, and phosphorescence. Several measuring modes are available, including wavelength scan, time scan, photometry and 3-D scan measurement. The spectrophotometer has sensitivity in the range of 50 picomoles of fluorescein when using a 300 &#956;L sample volume in the microplate, and is capable of measuring scan speeds of 60,000 nm/minute. It also has a wide dynamic range of up to 5 orders of magnitude which allows for the use of calibration curves over a wide range of concentrations. The optical system uses all reflective optics for maximum energy and sensitivity. The standard wavelength range is 200 to 750 nm, and can be extended to 900 nm when using one of the optional near infrared photomultipliers. The system allows optional temperature control for the plate reader from 5 to 60 degrees Celsius using an optional external temperature controlled liquid circulator. The microplate reader allows for the use of 96 well microplates, and the measuring speed for 96 wells is less than 60 seconds when using the kinetics mode.
Software controls for the F-7000 and Microplate Reader are also highly flexible. Samples may be set in either column or row formats, and any combination of wells may be chosen for sample measurements. This allows for optimal utilization of the microplate. Additionally, the software allows importing micro plate sample configurations created in Excel and saved in comma separated values, or "csv" format. Microplate measuring configurations can be saved and recalled by the software for convenience and increased productivity. Data results can be output to a standard report, to Excel, or to an optional Report Generator Program.

Demonstration of quantification of dsDNA using Molecular Probes PicoGreen dye and Hitachi F-7000 Fluorescence Spectrophotometer equipped with a microplate reader accessory.

Quantification of DNA, especially in small concentrations, is an important task with a wide range of biological applications including standard molecular ...

Absolute Quantum Yield Measurement of Powder Samples

Measurement of fluorescence quantum yield has become an important tool in the search for new solutions in the development, evaluation, quality control and research of illumination, AV equipment, organic EL material, films, filters and fluorescent probes for bio-industry.
Quantum yield is calculated as the ratio of the number of photons absorbed, to the number of photons emitted by a material. The higher the quantum yield, the better the efficiency of the fluorescent material.
For the measurements featured in this video, we will use the Hitachi F-7000 fluorescence spectrophotometer equipped with the Quantum Yield measuring accessory and Report Generator program. All the information provided applies to this system. 
Measurement of quantum yield in powder samples is performed following these steps:
<ol>
 <li>Generation of instrument correction factors for the excitation and emission monochromators. This is an important requirement for the correct measurement of quantum yield. It has been performed in advance for the full measurement range of the instrument and will not be shown in this video due to time limitations.</li>
 <li>Measurement of integrating sphere correction factors. The purpose of this step is to take into consideration reflectivity characteristics of the integrating sphere used for the measurements.</li>
 <li>Reference and Sample measurement using direct excitation and indirect excitation.</li>
 <li>Quantum Yield calculation using Direct and Indirect excitation. Direct excitation is when the sample is facing directly the excitation beam, which would be the normal measurement setup. However, because we use an integrating sphere, a portion of the emitted photons resulting from the sample fluorescence are reflected by the integrating sphere and will re-excite the sample, so we need to take into consideration indirect excitation. This is accomplished by measuring the sample placed in the port facing the emission monochromator, calculating indirect quantum yield and correcting the direct quantum yield calculation.</li>
 <li>Corrected quantum yield calculation.</li>
 <li>Chromaticity coordinates calculation using Report Generator program.</li>
</ol>
The Hitachi F-7000 Quantum Yield Measurement System offer advantages for this
application, as follows:
<ul>
 <li>High sensitivity (S/N ratio 800 or better RMS). Signal is the Raman band of water measured under the following conditions: Ex wavelength 350 nm, band pass Ex and Em 5 nm, response 2 sec), noise is measured at the maximum of the Raman peak. High sensitivity allows measurement of samples even with low quantum yield. Using this system we have measured quantum yields as low as 0.1 for a sample of salicylic acid and as high as 0.8 for a sample of magnesium tungstate.</li>
 <li>Highly accurate measurement with a dynamic range of 6 orders of magnitude allows for measurements of both sharp scattering peaks with high intensity, as well as broad fluorescence peaks of low intensity under the same conditions. </li>
 <li>High measuring throughput and reduced light exposure to the sample, due to a high scanning speed of up to 60,000 nm/minute and automatic shutter function. </li>
 <li>Measurement of quantum yield over a wide wavelength range from 240 to 800 nm.</li>
 <li>Accurate quantum yield measurements are the result of collecting instrument spectral response and integrating sphere correction factors before measuring the sample.</li>
 <li>Large selection of calculated parameters provided by dedicated and easy to use software.
</li>
</ul> 
During this video we will measure sodium salicylate in powder form which is known to have a quantum yield value of 0.4 to 0.5.

In this video we will demonstrate measuring and calculating absolute quantum yield and chromaticity coordinates directly in powder samples using the Hitachi F-7000 Quantum Yield Measuring System.

Measurement of fluorescence quantum yield has become an important tool in the search for new solutions in the development, evaluation, quality control and ...

Rapid Fibroblast Removal from High Density Human Embryonic Stem Cell Cultures

Mouse embryonic fibroblasts (MEFs) were used to establish human embryonic stem cells (hESCs) cultures after blastocyst isolation1. This feeder system maintains hESCs from undergoing spontaneous differentiation during cell expansion. However, this co-culture method is labor intensive, requires highly trained personnel, and yields low hESC purity4. Many laboratories have attempted to minimize the number of feeder cells in hESC cultures (i.e. incorporating matrix-coated dishes or other feeder cell types5-8). These modified culture systems have shown some promise, but have not supplanted the standard method for culturing hESCs with mitomycin C-treated mouse embyronic fibroblasts in order to retard unwanted spontaneous differentiation of the hESC cultures. Therefore, the feeder cells used in hESC expansion should be removed during differentiation experiments. Although several techniques are available for purifying the hESC colonies (FACS, MACS, or use of drug resistant vectors) from feeders, these techniques are labor intensive, costly and/or destructive to the hESC. The aim of this project was to invent a method of purification that enables the harvesting of a purer population of hESCs. We have observed that in a confluent hESC culture, the MEF population can be removed using a simple and rapid aspiration of the MEF sheet. This removal is dependent on several factors, including lateral cell-to-cell binding of MEFs that have a lower binding affinity to the styrene culture dish, and the ability of the stem cell colonies to push the fibroblasts outward during the generation of their own "niche". The hESC were then examined for SSEA-4, Oct3/4 and Tra 1-81 expression up to 10 days after MEF removal to ensure maintenance of pluripotency. Moreover, hESC colonies were able to continue growing from into larger formations after MEF removal, providing an additional level of hESC expansion.

Despite ongoing efforts to transition cultures to feeder-free conditions, the derivation and culture of human embryonic stem cells (hESC) remain largely dependent on co-cultures with mouse embryonic feeders (MEFs). Here, we show a novel methodology for rapidly removing feeders from hESC cultures prior to experimentation.

Mouse embryonic fibroblasts (MEFs) were used to establish human embryonic stem cells (hESCs) cultures after blastocyst isolation1. This feeder system ...

Probing High-density Functional Protein Microarrays to Detect Protein-protein Interactions

High-density functional protein microarrays containing ~4,200 recombinant yeast proteins are examined for kinase protein-protein interactions using an affinity purified yeast kinase fusion protein containing a V5-epitope tag for read-out. Purified kinase is obtained through culture of a yeast strain optimized for high copy protein production harboring a plasmid containing a Kinase-V5 fusion construct under a GAL inducible promoter. The yeast is grown in restrictive media with a neutral carbon source for 6 hr followed by induction with 2% galactose. Next, the culture is harvested and kinase is purified using standard affinity chromatographic techniques to obtain a highly purified protein kinase for use in the assay. The purified kinase is diluted with kinase buffer to an appropriate range for the assay and the protein microarrays are blocked prior to hybridization with the protein microarray. After the hybridization, the arrays are probed with monoclonal V5 antibody to identify proteins bound by the kinase-V5 protein. Finally, the arrays are scanned using a standard microarray scanner, and data is extracted for downstream informatics analysis1,2 to determine a high confidence set of protein interactions for downstream validation in vivo.

Using protein microarrays containing nearly the entire S. cerevisiae proteome is probed for rapid unbiased interrogation of thousands of protein-protein interactions in parallel. This method can be utilized for protein-small molecule, posttranslational modification, and other assays in high-throughput.

High-density functional protein microarrays containing ~4,200 recombinant yeast proteins are examined for kinase protein-protein interactions using an ...

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 ...

High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays focus on transcriptional regulation, being essentially aimed at the identification of promoter-targeting molecules. As a result, post-transcriptional mechanisms are largely uncovered by gene expression targeted drug development. Here we describe a cell-based assay aimed at investigating the role of the 3&#39; untranslated region (3&#8217; UTR) in the modulation of the fate of its mRNA, and at identifying compounds able to modify it. The assay is based on the use of a luciferase reporter construct containing the 3&#8217; UTR of a gene of interest stably integrated into a disease-relevant cell line. The protocol is divided into two parts, with the initial focus on the primary screening aimed at the identification of molecules affecting luciferase activity after 24 hr of treatment. The second part of the protocol describes the counter-screening necessary to discriminate compounds modulating luciferase activity specifically through the 3&#8217; UTR. In addition to the detailed protocol and representative results, we provide important considerations about the assay development and the validation of the hit(s) on the endogenous target. The described cell-based reporter gene assay will allow scientists to identify molecules modulating protein levels via post-transcriptional mechanisms dependent on a 3&#8217; UTR.

Here we describe a cell-based reporter gene assay as a valuable tool to screen chemical libraries for compounds modulating post-transcriptional control mechanisms exerted through 3&#8217; UTR.

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays ...

Detection of miRNA Targets in High-throughput Using the 3'LIFE Assay

Luminescent Identification of Functional Elements in 3&#8217;UTRs (3&#8217;LIFE) allows the rapid identification of targets of specific miRNAs within an array of hundreds of queried 3&#8217;UTRs. Target identification is based on the dual-luciferase assay, which detects binding at the mRNA level by measuring translational output, giving a functional readout of miRNA targeting. 3&#8217;LIFE uses non-proprietary buffers and reagents, and publically available reporter libraries, making genome-wide screens feasible and cost-effective. 3&#8217;LIFE can be performed either in a standard lab setting or scaled up using liquid handling robots and other high-throughput instrumentation. We illustrate the approach using a dataset of human 3&#8217;UTRs cloned in 96-well plates, and two test miRNAs, let-7c and miR-10b. We demonstrate how to perform DNA preparation, transfection, cell culture and luciferase assays in 96-well format, and provide tools for data analysis. In conclusion 3&#39;LIFE is highly reproducible, rapid, systematic, and identifies high confidence targets.

Luminescent identification of functional elements in 3&#8217; untranslated regions (3&#8217;UTRs) (3&#8217;LIFE) is a technique to identify functional regulation in 3&#8217;UTRs by miRNAs or other regulatory factors. This protocol utilizes high-throughput methodology such as 96-well transfection and luciferase assays to screen hundreds of putative interactions for functional repression.

Luminescent Identification of Functional Elements in 3&#8217;UTRs (3&#8217;LIFE) allows the rapid identification of targets of specific miRNAs within an ...

Isolation of Mitochondria from Minimal Quantities of Mouse Skeletal Muscle for High Throughput Microplate Respiratory Measurements

Dysfunctional skeletal muscle mitochondria play a role in altered metabolism observed with aging, obesity and Type II diabetes. Mitochondrial respirometric assays from isolated mitochondrial preparations allow for the assessment of mitochondrial function, as well as determination of the mechanism(s) of action of drugs and proteins that modulate metabolism. Current isolation procedures often require large quantities of tissue to yield high quality mitochondria necessary for respirometric assays. The methods presented herein describe how high quality purified mitochondria (~ 450 &#181;g) can be isolated from minimal quantities (~75-100 mg) of mouse skeletal muscle for use in high throughput respiratory measurements. We determined that our isolation method yields 92.5&#177; 2.0% intact mitochondria by measuring citrate synthase activity spectrophotometrically. In addition, Western blot analysis in isolated mitochondria resulted in the faint expression of the cytosolic protein, GAPDH, and the robust expression of the mitochondrial protein, COXIV. The absence of a prominent GAPDH band in the isolated mitochondria is indicative of little contamination from non-mitochondrial sources during the isolation procedure. Most importantly, the measurement of O2 consumption rate with micro-plate based technology and determining the respiratory control ratio (RCR) for coupled respirometric assays shows highly coupled (RCR; &#62;6 for all assays) and functional mitochondria. In conclusion, the addition of a separate mincing step and significantly reducing motor driven homogenization speed of a previously reported method has allowed the isolation of high quality and purified mitochondria from smaller quantities of mouse skeletal muscle that results in highly coupled mitochondria that respire with high function during microplate based respirometirc assays.

Here, we present a modification of a previously reported method that allows for the isolation of high quality and purified mitochondria from smaller quantities of mouse skeletal muscle. This procedure results in highly coupled mitochondria that respire with high function during microplate based respirometirc assays.

Dysfunctional skeletal muscle mitochondria play a role in altered metabolism observed with aging, obesity and Type II diabetes. Mitochondrial ...

Isolation of Intact Mitochondria from Skeletal Muscle by Differential Centrifugation for High-resolution Respirometry Measurements

Mitochondria are involved in cellular energy metabolism and use oxygen to produce energy in the form of adenosine triphosphate (ATP). Differential centrifugation at low- and high-speed is commonly used to isolate mitochondria from tissues and cultured cells. Crude mitochondrial fractions obtained by differential centrifugation are used for respirometry measurements. The differential centrifugation technique is based on the separation of organelles according to their size and sedimentation velocity. The isolation of mitochondria is performed immediately after tissue harvesting. The tissue is immersed in an ice-cold homogenization medium, minced using scissors and homogenized in a glass homogenizer with a loose-fitting pestle. The differential centrifugation technique is efficient, fast and inexpensive and the mitochondria obtained by differential centrifugation are pure enough for respirometry assays. Some of the limitations and disadvantages of isolated mitochondria, based on differential centrifugation, are that the mitochondria can be damaged during the homogenization and isolation procedure and that large amounts of the tissue biopsy or cultured cells are required for the mitochondrial isolation.

Here, a quadriceps muscle specimen is taken from an anaesthetized pig and mitochondria are isolated by differential centrifugation. Then, the respiratory rates of mitochondrial respiratory chain complexes I, II and IV are determined using high-resolution respirometry.