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.采集血样<ol><li>收集空腹外周静脉血样品到含有通过在肘窝的一个突出静脉标准静脉穿刺抗凝乙二胺四乙酸（EDTA）（其比其它抗凝剂有几个优点）在10毫升的塑料管中。 </li><li>离心血液样品在1600×g离心20分钟，在4℃下在台式离心机，以获得自由的红血细胞和少量的RNA的等离子体。 </li><li>依次离心上清在吊桶式转子3000克（4℃）10分钟，以除去白细胞和血小板，然后附加15分钟以分别除去残留的细胞碎片。 </li><li>用在RT密度计根据生产指令测量等离子体的密度。 注:密度调节（D =1.023克/ ml）的0.9％生理盐水溶液可以去除外来的，但之前的密度梯度超速离心后是必需的。 </li></OL&gt; 2.从血浆中去除的外来体<ol><li>删除循环外来体具有类似高密度脂蛋白的密度，代表的miRNA 3的定量显著来源。 <ol><li>通过加入252微升外切体沉淀溶液至1毫升血浆温育30分钟，在4℃下执行此操作。以沉淀出外来体，离心混合物30分钟，在4℃1500克。 </li><li>以分离的HDL，将所得上清转移1ml至聚碳酸酯厚壁超速离心管，以便进一步处理用密度梯度超速离心（见下文）。 </li></ol></li></ol> 3.密度梯度超速离心（图1）。 <ol><li>到单独的HDL使用采用具有一个固定角转子以448811×G下8℃，分别操作和地板超速离心机3步骤的过程。 </li><li>准备了三种不同密度解决方案顺序和新鲜的每个隔离。 <ol><li>制备溶液A（VLDL的隔离峰，d = 1.006克/毫升）将11.4克NaCl（氯化钠:0.195摩尔）1000毫升高压灭菌蒸馏水，0.1g的EDTA2Na和1ml 1N NaOH中。然后添加一个额外的3毫升蒸压蒸水。 </li><li>准备溶液B（LDL隔离，D =1.182克/毫升）加入25.2克溴化钠至100毫升溶液A（氯化钠0.195摩尔，溴化钠2.44摩尔）。 </li><li>通过混合78.8克溴化钠用100毫升的溶液A（氯化钠0.195摩尔，溴化钠7.7摩尔）准备论文C（高密度脂蛋白的隔离，D =1.470克/毫升）。确认在室温下用密度适当的密度。保留所有的解决方案在4°C，直到进一步使用。 </li></ol></li></ol> 4.隔离的VLDL <ol><li>混合1毫升血浆（平均密度= 1.023克/毫升）和无核酸酶的200微升发红色7B的在6.5毫升聚碳酸酯厚壁超速离心管。 </li><li>然后小心层上的混合物前5 ml溶液A的。如果需要，在求解的T顶部增加额外的脂肪红7B直径:平衡每个管的重量。离心2小时（加速度 - 5），（减速 - 7）。 注:在离心分离中，脂蛋白被累积作为它们的平衡密度区域的频带。 </li><li>在运行的端部观察2层。除去1.5毫升代表在4℃的顶层和商店的VLDL级分。 </li><li>最后，用移液管转移从管的含有低密度脂蛋白的底面4毫升，高密度脂蛋白，白蛋白和脂肪酸馏分为LDL隔离一个新的聚碳酸酯管。 </li></ol> 5.隔离的LDL <ol><li>混合溶液2ml B和100微升不含核酸酶的脂肪红7B的成含有LDL和HDL级分（第4部分）的管，分别。 </li><li>然后离心进行3小时（加速9，减速7）。此后，除去1.5毫升代表顶层LDL部分的，并保持在4℃或储存在-80℃。最后，从管的含有高密度脂蛋白的底部转移4毫升部分，以一个新的聚碳酸酯管。 </li></ol> 6.隔离的HDL <ol><li>混合溶液2ml的C，100微升不含核酸酶的脂肪红7B和15微升98％β巯基乙醇的到含有HDL馏分分别在管。 </li><li>离心3小时（加速9，减速7）。然后除去2-毫升HDL馏分表示顶层，要么保持在4℃或储存在-80℃。 </li></ol> 7.脱盐和脂蛋白成分浓度<ol><li>以避免与随后的琼脂糖凝胶电泳和PCR的干扰，删除使用由制造商的说明用适当的分子量截留（3K管VLDL和10K管的LDL / HDL）的离心过滤装置密度梯度超速离心期间加入过量的盐。 <ol><li>简言之，加入2.5毫升冷的PBS（137 mM氯化钠，2.7毫KCL，8毫磷酸氢二钠，2毫KH 2 PO 4; pH 7.4）中后centrifu戈整个VLDL级分收集，在密度梯度超离心在4℃下使用吊桶式转子60分钟。 </li><li>用10ml冰冷的PBS脱盐LDL部分两次，每次30分钟。接着，使用13毫升冰冷的PBS两次脱盐HDL级分。较高的PBS体积是必要改善与琼脂糖凝胶电泳迁移率。离心后，取出含有溶质脂蛋白，并保持在4℃或储存在-80℃。 </li></ol></li></ol> 8.琼脂糖凝胶电泳<ol><li>亲热采用与制造商的说明稍作修改如下所述试剂盒脂蛋白琼脂糖凝胶电泳。 注:该步骤是只评估浓缩脂蛋白样品的质量和纯度。 <ol><li>简言之，获得6微升脱盐脂蛋白馏分与密度梯度超速离心，并加载到预浇铸脂蛋白凝胶。使用人类lipoprotein标准VLDL，低密度脂蛋白和高密度脂蛋白的尺寸参考。进行电泳在RT在100V供使用Rep准备缓冲器60分钟。 </li><li>干燥凝胶10分钟，然后染色在室温10分钟以脂肪红7B。脱色的甲醇 - 水75:25（体积/体积）和干燥的5分钟的混合物再次凝胶。 </li></ol></li></ol> 9. RNA提取与纯化<ol><li>使用血清/血浆的miRNA分离和纯化试剂盒由纯化的人HDL亲热的miRNA的隔离。 <ol><li>简言之，加入1ml RNA裂解试剂200微升纯化的高密度脂蛋白，用涡旋混合，然后在室温下孵育5分钟以确保核蛋白复合物和RNA酶失活的完全分离。 </li><li>然后穗3.5微升合成线虫的microRNA（CEL-的miR-39; 1.6×10 8个拷贝/微升）到混合物中。然后根据制造商的说明书进行RNA提取。 </li></ol></li><li>执行purificati上提取的体miRNA与洗脱旋转柱按照生产商的说明。测量的miRNA从与spectrophorometer纯化的高密度脂蛋白的浓度。 注意:从自旋列的miRNA的洗脱使用16微升的无RNase水。 </li></ol> 10.反转录（RT-PCR） <ol><li>隔离100ng的所述miRNA从HDL的掺有合成的miRNA（CEL-的miR-39），并在使用逆转录试剂盒，并根据制造商的说明20微升反应体积逆转录。 </li><li>没有模板的miRNA（NTC）和无逆转录酶混合物（NRT）进行适当的控制。 </li></ol> 11.实时PCR（定量RT-PCR） <ol><li> 2稀释的cDNA，10微升PCR混合物，2微升的通用引物，2微升的miRNA的引物和4微升不含RNA酶的水:在20微升用2微升1的总体积亲热实时PCR。 </li><li>运行reacti对在95℃下15分钟，96孔板，随后为15秒和55℃30秒和在70℃的延伸相对于30 45个循环的94℃s.ec亲热一式三份所有反应。 </li><li>接着，的miRNA Calcuate相对量通过使用2-ΔΔ的Ct方法正常化到合成持家基因按制造商的说明之后。 </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. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Separation+of+plasma+lipoproteins+by+density-gradient+ultracentrifugation.">Separation of plasma lipoproteins by density-gradient ultracentrifugation.</a> Anal Biochem. 65, 42-49 (1975).</li><li>Foreman, J. R., 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=Fractionation+of+human+serum+lipoproteins+by+single-spin+gradient+ultracentrifugation:+quantification+of+apolipoproteins+B+and+A-1+and+lipid+components.">Fractionation of human serum lipoproteins by single-spin gradient ultracentrifugation: quantification of apolipoproteins B and A-1 and lipid components.</a> J Lipid Res. 18, 759-767 (1977).</li><li>Dong, 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=Serum+LDL-+and+HDL-cholesterol+determined+by+ultracentrifugation+and+HPLC.">Serum LDL- and HDL-cholesterol determined by ultracentrifugation and HPLC.</a> J Lipid Res. 52, 383-388 (2011).</li><li>Tong, H., Knapp, H. R., VanRollings, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=low+temperature+flotation+method+to+rapidly+isolate+lipoproteins+from+plasma.">low temperature flotation method to rapidly isolate lipoproteins from plasma.</a> J Lipid Res. 39, 1696-1704 (1998).</li><li>Fless, G. M., ZumMallen, M. E., Scanu, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physicochemical+properties+of+apolipoprotein+(a)+and+lipoprotein+(a-)+derived+from+the+dissociation+of+human+plasma+lipoprotein+(a).">Physicochemical properties of apolipoprotein (a) and lipoprotein (a-) derived from the dissociation of human plasma lipoprotein (a).</a> J Biol Chem. 261, 8712-8718 (1986).</li><li>Brownie, 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=The+elimination+of+primer-dimer+accumulation+in+PCR.">The elimination of primer-dimer accumulation in PCR.</a> Nucleic Acids Res. 25, 3235-3241 (1997).</li><li>Alton, E., Inyoul, L., Leroy, H., David, G., Kai, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+microRNA:+a+new+source+of+biomarkers.">Extracellular microRNA: a new source of biomarkers.</a> Mutat Res. 717 (1-2), 85-90 (2011).</li><li>Stefanie, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+biomarker+profiling+with+microRNAs.">Cancer biomarker profiling with microRNAs.</a> Nature Biotechnology. 26, 400-401 (2008).</li><li>Prasun, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNAs+as+promising+biomarkers+in+cancer+diagnostics.">MicroRNAs as promising biomarkers in cancer diagnostics.</a> Biomarker Research. 2 (19), (2014).</li><li>Creemers, E. E., Tijsen, A. J., Pinto, Y. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circulating+microRNAs:+novel+biomarkers+and+extracellular+communicators+in+cardiovascular+disease?.">Circulating microRNAs: novel biomarkers and extracellular communicators in cardiovascular disease?.</a> Circ Res. 110, 483-495 (2012).</li><li>Jonathan, S., Martin, S., Eric, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> miRNA profiling from blood -challenges and recommendations. , (2015).</li><li>Francesco, M., Paola, D. C., Anna, T., Jesper, T., Sergio, A., Riccardo, L. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Normalization+of+circulating+microRNA+expression+data+obtained+by+quantitative+real-time+RT-PCR.">Normalization of circulating microRNA expression data obtained by quantitative real-time RT-PCR.</a> Brief Bioinform. 3, 1-9 (2015).</li><li>Chen, Y., 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=Circulating+microRNAs,+novel+biomarkers+of+acute+myocardial+infarction:+a+systemic+review.">Circulating microRNAs, novel biomarkers of acute myocardial infarction: a systemic review.</a> World J Emerg Med. 3, 257-260 (2012).</li></ol>

作者什么都没有透露。

从血液新型生物标志物的鉴定将在各种疾病的临床诊断和预后提供帮助。微RNA已经已知具有生物标志物的所有品质，并在各种研究14-17都被显示。在这项研究中，我们已经证明快速，简便容易的方法从血浆HDL隔离的miRNA。 VLDL，LDL和HDL的隔离的常规密度梯度超离心方法取决于等离子体的精确采样，缓冲溶液的精确配制，密度和底部脂蛋白馏分8的定量转移的测量。有已经描述分离的HD...

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.

高密度脂蛋白的分离外来体切除后 从高度纯化的HDL获得miRNA的有必要删除代表的miRNA污染7的来源的外来体。这是之前的密度梯度超速离心用市售的试剂盒进行。为了实用目的由商业公司开发的三步标准密度梯度超速离心协议被修改（ 图1）。此协议需要与140000 rpm的速度的固定角转子，其比用离心力常用协议基本上快高达5...

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

微RNA起重要的调节作用，并正在成为关于各种人类疾病新的治疗目标。它已经显示，微RNA在高密度脂蛋白携带。我们已经开发出一种简化方法迅速隔离适用于从人血浆中的miRNA分析纯化HDL。

从高纯高密度脂蛋白的miRNA的高通量快速分离和简化方法

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

11.8K 次观看.  Virginia Commonwealth University. 微RNA起重要的调节作用，并正在成为关于各种人类疾病新的治疗目标。它已经显示，微RNA在高密度脂蛋白携带。我们已经开发出一种简化方法迅速隔离适用于从人血浆中的miRNA分析纯化HDL。 

视频: 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.

一个神经元和星形胶质细胞共培养检测神经毒性的高含量分析

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

科研

生物学

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.

双链DNA定量，使用日立F - 7000荧光分光光度计和PicoGreen染料

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.

粉末样品的绝对量子产量测量

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.

快速高密度的人类胚胎干细胞培养成纤维细胞去除

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.

探讨高密度功能蛋白微阵列来检测蛋白质 - 蛋白质相互作用

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.

小RNA分子的高效结扎的微小RNA定量高通量测序

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.

高通量筛选转录后调控基因的化学调节剂

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.

检测miRNA靶在高通量的使用3&#39;LIFE分析

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.

从小鼠骨骼肌高通量微孔板呼吸测量最低数量的隔离线粒体

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.