Name: fast and simplified method for high through-put isolation of mirna from highly purified high density lipoprotein
Uploaded: 2016-07-27T13:00:00.000+00:00
Duration: 9 min 48 s
Description: Watch this Scientific Journal Video about Fast and Simplified Method for High Through-put Isolation of miRNA from Highly Purified High Density Lipoprotein at JoVE.com

This work was supported, in whole or in part, by NIH Grants R01 AA 020758-04, U01DK 061731-13 and T32 DK 007150-38 to AJS and T32 DK 007150-38 to AA. This is original work and is not under consideration elsewhere for publication.

1.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>

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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HDL 8 -11的许多方法。这些方法往往不是很费力，或需要大量血浆和透析广泛用于脱盐分离的脂蛋白，不彻底清除外来体和脂蛋白作为的miRNA 3的来源。为了解决我们建立这个SI这些缺点mple和血浆HDL的miRNA隔离的快捷方法。 的主要目标和本研究的主要目的是，以分离和无红细胞通过超速离心分离的HDL-miRNA的络合物，血沉棕黄层，外来体，分泌囊泡，VLDL，LDL和脂蛋白（一）的干扰，然后分离从纯化的HDL的miRNA。任何这些组分与HDL的污染和干扰将改变预期的实验结果和miRNA分析数据。从血液的DNA，RNA和微RNA隔离是一个非常困难的任务，由于其复杂性。它一直提出用很多核酸（包括微RNA）提取和纯化相比细胞，组织和器官样品18的技术挑战。也有存在于循环血液细胞中相对较少的miRNA基因。血是外来体，分泌泡和配有免费循环miRNA的高密度脂蛋白一起，这是相对较少18已知载体。因此，大的血浆样品体积的安装需要的HDL-miRNA的复杂的处理和分离。此外，miRNA及其靶序列，短性质使得它更难以实现足够的特异性以标准的PCR寡核苷酸的技术。血液的细胞的组件总是知道分泌的miRNA和mRNA响应于外部环境包括温度，微生物和毒素或应力发生变化。此外核酸酶，RNA酶，蛋白质循环和其他酶血液中存在将影响这些miRNA的隔离。 同时采用实时定量RT-PCR的方法ΔΔCT循环miRNA的放大也缺乏行之有效的知名看家基因β一样，肌动蛋白或GAPDH数据正常化。这再次提出了从miRNA的血液循环血清或血浆19分析的主要障碍之一。常用的短期非编码52]形成和snRNAs在血液和该M很少表达在碟刹利用它们作为内部控制基因的进一步困难。解决52]形成和snRNAs的这些缺点，并避免我们使用血清/血浆合成穗以控制在测定作为内部对照基因作为根据制造商的协议遵循任何技术上的困难。它有助于在正常化为任何非特异性扩增和miRNA纯化和PCR反应18期间发生的变化。即使是在miRNA的退化或不存在任何特定的miRNA放大信号的情况下，在穗中控制在定量RT-PCR反应应扩增，并与合理恒定和均匀的Ct值给一个预期信号。基于三阴性对照和阳性对照沿控制放大秒杀，我们得到了的miR-223基因好最佳的数据验证。这证实了我们已经得到的miR-223的良好的收益率从这种简单的方法循环血浆高密度脂蛋白。 总之，我们已经建立了一个方法○˚F密度梯度超速离心（DGUC），增加β巯基乙醇的从血浆HDL的miRNA纯化。该方法具有几个优点:（a）VLDL，LDL和HDL由地板超速离心机的时间很短的时间内分离比较需要24的其它方法- 96小时8-11; （二）低密度脂蛋白和高密度脂蛋白透析，脱盐/浓缩，取1小时内发生与花24个小时以上8,9等方法进行了比较; （三）脂蛋白的污染是由HDL馏分β巯基乙醇除去，但并不意在LDL部分中​​使用; （四）所需要的样本量仅为1毫升所有逐步VLDL，低密度脂蛋白和高密度脂蛋白隔离与需要更多的样本量等方法进行比较; （五）隔离11中最小的氧化损伤HDL; （F）的12个样品可以在单一脂蛋白分离分析运行中进行分析; （G）提取HDL的250微升样本量足够分析米核糖核酸浓度/纯度，琼脂糖凝胶电泳，蛋白质浓度，微阵列和RT-qPCR的程序。我们的方法的局限性是，HDL-miRNA的小百分比可以在外来体沉淀步骤失去和所需的血浆样品的体积应超过200微升以分离的HDL-miRNA的。 最后，将等离子体微RNA不仅从损坏或叛徒血细胞中的循环，而且从健康正常的血细胞和从主体，其包括受体正在进行的健康状况的各种健康和患病的组织和器官的其他部分细胞的20.目前这个研究系统地从人血浆中分离的高密度脂蛋白纯化成熟miR-223。这项研究清楚地表明，高纯度的高密度脂蛋白血浆中成熟miR-223水平检测，稳定，重复性好，个人之间的一致的样本，从而大大促进了脂蛋白，李临床使用的未来性测试版本，心血管疾病等代谢综合征。令人惊奇的是，成熟miRNA，特别的miR-223从HDL等离子体可能是耐核酸酶消化等恶劣条件，可能解释在纯化的高密度脂蛋白的miR-223的稳定性。的miR-223的抗RNA酶的机制需要进一步研究。显然，在这些纯化的血浆HDL研究的miRNA表达谱将在研究各种弊端对未来的miRNA标志线索。等离子体中的这些高密度脂蛋白相关微RNA可以用作潜在的临床诊断的生物标志物和在将来个性化医疗。

MicroRNAs are endogenous non-coding tiny RNA species that are highly conserved and are considered key players in the regulation of various biological processes by degrading or repressing specific target messenger RNAs1. Because miRNAs act intracellularly they have been explored as tissue-derived biomarkers which led to the discovery of tissue-specific functions of these miRNA. However, miRNAs are also found extracellularly either associated with proteins or in exosomes/micro vesicles that effectively can shield them from degradation by extracellular RNases2. More recent studies have shown that the protective effect of HDL may not be closely linked to its capability to promote cholesterol efflux but rather to its non-cholesterol cargo, in particularly as a circulating miRNAs carrier 3, 4. These miRNAs may not only modulate lipid metabolism but are also associated with anti-inflammatory, antioxidant and antithrombotic effects of the HDL-miRNA complex 5, 6.
To further explore the role of miRNAs carried in HDL particles, a simple and easy protocol needs to be established for miRNA extraction from isolated highly purified HDL for use in clinical routine. Numerous methods have been described to isolate HDL. These methods are either very time consuming or require large volume of plasma that may require sample pooling, extensive dialysis for desalting isolated lipoproteins and they do not completely remove exosomes as a source of miRNAs3, respectively. Here we describe a simple and rapid method that can isolate miRNA from highly purified HDL utilizing small volume of blood samples on a larger scale. We believe that this method may serve as good reference to promote research into the role of circulating miRNAs and in particular the role of HDL in facilitating communication between various cells and organs.

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

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

Small non-coding RNAs (miRNAs) have been implicated in a variety of human diseases including metabolic syndromes. They may be utilized as biomarkers for diagnosis and prognosis or may serve as targets for drug development, respectively. Recently it has been shown that miRNAs are carried in lipoproteins, particularly high density lipoproteins (HDL) and are delivered to recipient cells for uptake. This raises the possibility that miRNAs play a critical and pivotal role in cellular and organ function via regulation of gene expression as well as messenger for cell-cell communications and crosstalk between organs. Current methods for miRNA isolation from purified HDL are impractical when utilizing small samples on a large scale. This is largely due to the time consuming and laborious methods used for lipoprotein isolation. We have developed a simplified approach to rapidly isolate purified HDL suitable for miRNA analysis from plasma samples. This method should facilitate investigations into the role of miRNAs in health and disease and in particular provide new insights into the variety of biological functions, outside of the reverse cholesterol transport, that have been ascribed to HDL. Also, the miRNA species which are present in HDL can provide valuable information of clinical biomarkers for diagnosis of various diseases.

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54,000转3，8，9，和10离心。雇用一个广泛使用的T-1270的转子具有70000转的最大力量，离心最初是与未能抵抗离心力聚异试管中进行。为了避免管的崩溃，已成功应用于聚碳酸酯管。离心时间为脂蛋白的氧化和可能的miRNA降解11是至关重要的。因此，几个不同的离心时间，从一共有8至96小时的测试。分别基于离心时间和力量，在该离心分离进行另外温度调节。 高密度脂蛋白成分的纯度 分离的高密度脂蛋白馏分的纯度用琼脂糖凝胶电泳进行了检查。 图2.示出了用于由梯度超速离心分离出的高密度脂蛋白减法典型电泳结果。这清楚地表明，HDL是缺乏VLDL的任何污染，并呈现出典型的α-流动性。缺乏在VLDL和LDL馏分任何α-迁移脂蛋白的演示ultracent期间HDL完全恢复rifugation一步。将HDL组分，然而，也表现出β-流动性，一种已知的电泳带型的一些痕量由于与脂蛋白污染（脂蛋白（a））12。 从隔离HDL脂蛋白（a）消除 留学HDL携带的miRNA需要纯度最高的HDL的隔离。 Lp的干扰（a）用高密度脂蛋白可能有助于高密度脂蛋白的交叉污染，在低密度脂蛋白携带的miRNA。为了最大限度地减少的HDL的这种污染，β巯基乙醇10 15微升在最后离心步骤（ 图1）加到溶液C。 β巯基乙醇的加成已显示未改变的HDL密度属性10。如显示于图3，除了β巯基乙醇的导致在不存在具有非常有效地除去脂蛋白（a）的一致的任何B-迁移脂蛋白。 的miRNA纯化HDL miRNA的分离最初尝试其通常用于从血液中的RNA的提取采用裂解试剂。虽然这种方法产生了非常好的RNA产量（91.45纳克/微升），但经过分光光度分析表明不尽人意RNA的纯度。通过除去苯酚的分离的RNA的额外纯化改善了纯度，但得到的RNA量现在显著低。接着，另一试剂盒测试其显示可接受的RNA的纯度（1.7 260/280纳米比），但的RNA产率26倍下与裂解试剂（3.45 与 91.45毫微克/微升）比较。为可接受的RNA的纯度以良好的产率的最佳结果与miRNeasy血清/血浆试剂盒获得（48.2毫微克/微升; 1.6 260/280纳米比），因此，该提取步骤，随后从所述分离的用于检测的miRNA HDL脂蛋白分数。 3的货物识别，并且显示出抑制高密度脂蛋白胆固醇摄取5。在图4A中 。显示扩增曲线的的miR-223 PCR反应和优化后的比较基准阈值和循环阈值的典型日志情节飙升，在CEL-MIR-39。该合成基因被用作内部对照，因为没有在等离子体对miRNA的已知归一化控制的报告。此外，几个潜在的确立的内源性管家基因像RNU6-2，RNU-48，HY3无法检测和SNORD95可以以纯化的HDL被检测到，但是，在的miR-223和SNORD95的Ct值的差小于五（数据未示出）。 如图5所示熔解曲线分析清楚地显示了一个不同的单一峰与在前述的PCR选择性的miRNA的扩增是一致的。如在图4B中。示出，这两个的miR-223和参考CEL-的mir-39可以一致地被在所有六个亲频带进行检测。观察了CEL-MIR-39的所有个体间差异相对较小用miR-223相比。这些发现支持高密度脂蛋白的分离高纯度允许其miRNA的货物的检测。 在纯化HDL的miR-223的检测 实时采用荧光定量PCR方法来量化双发夹结构的miRNA前体为单链的miRNA。此方法需要一个正向/反向基因特异性引物和热稳定的反转录酶与miRNA的发夹结构转换成cDNA。该cDNA随后被扩增，并使用实时定量PCR用SYBR绿检测的帮助下进行定量。从血清，血浆和纯化的HDL等离子体微RNA可以精确PROFIL采用ED miRNA的RT-PCR系统。 我们平均的miR-223基因表达的实验性产品纯化的HDL显示，30.9 Ct值及其相应的阴性对照上述截止35（NTC = 38.1 Ct值，NRT和NAC没有放大）。在这个实验中，我们将纯化的HDL样品中看到了引物-二聚体的形成具有低熔融温度（73℃中的miR-223和75.5℃的CEL-的miR-39）和比特定miRNA的测定更高（C T）值（ 表1）。 表1中的引物-二聚体可能发生，因为在溶液中的引物浓度高的。当引物分子30 PCR循环13日后彼此连接出现这种情况居多。这使他们能够退火到其它的引物分子，并在效果，成为线性迷你模板和它会导致更高的背景，并可能导致一个代（C T）值&lt;40的NTC的（没有template控制）样品。 <img alt="图1" src="/files/ftp_upload/54257/54257fig1.jpg" /> 图1.高密度脂蛋白的分离过程的示意图 。高密度脂蛋白是在一系列的三个离心步骤来制备密度梯度超速离心。脂蛋白带和中间馏分中的密度梯度分布所示。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54257/54257fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/54257/54257fig2.jpg" /> 脂蛋白隔离没有β巯基乙醇的 图2. 琼脂糖凝胶电泳。VLDL，通过密度梯度超速离心分离LDL和HDL的级分不添加β巯基乙醇至溶液C demonst评价脂蛋白（a）中的高密度脂蛋白馏分的存在。泳道1,2，9和10分别表示大小标记;车道3/4，5/6和7/8分别代表VLDL，低密度脂蛋白和高密度脂蛋白。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54257/54257fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/54257/54257fig3.jpg" /> HDL以β巯基乙醇隔离。最后离心步骤之前添加β巯基乙醇至溶液C 的 图3. 琼脂糖凝胶电泳导致从HDL级分中除去脂蛋白（a）的。泳道1,2，9和10分别表示大小标记;泳道3-8代表高密度脂蛋白。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54257/54257fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 图4.两种不同的miRNA扩增曲线和CEL-的miR-39和miR-223的表达。 （A）实时定量PCR采用隔离的HDL描述进行。 PCR反应45个循环，该曲线相交的阈值是（C T） -数值点运行。数据显示的表达CEL-的miR-39（左面板）和miR-223（右面板），分别。水平线表示检测阈值。一个35人的循环数被设置为截止为阳性扩增。（B）从6个样品获得的分离的HDL代表实时PCR数据被示出。生平均Ct值示为CEL-的miR-39和miR-223，其表达水平变化中纯化的高密度脂蛋白的血浆级分6个样品，每个之间从不同的供体不足2 Ct值。使用PCR系统和人血清和等离子MIRN进行表达谱一个定量RT-PCR。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54257/54257fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/54257/54257fig5.jpg" /> 图5.熔解曲线为CEL-的miR-39和miR-223。如图所示，一个单一的峰的存在，切尔的miR-39分别（右图）和miR-223（左面板），表示的特异性扩增。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54257/54257fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="表格1" src="/files/ftp_upload/54257/54257table1.jpg" /> 合成CEL-的miR-39的阴性和阳性对照和miR-223，其特征在血清，血浆，cDNA和纯化的高密度脂蛋白馏分的表1中 。行（C T）值。 NTC（无template控制），NRT（无逆转录酶），NAC（无放大控制，只定量RT-PCR的水和试剂）。

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 ...

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Nanyang Technological University

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JoVE Journal

Biology

11.9K 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

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

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

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

高密度脂蛋白的分离的非编码小RNA定量

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

科研

生物化学

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

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

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

高通量微RNA分析：优化复纳升秤定量RT - PCR Fluidig​​m公司动态ArrayTM国际金融中心

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

生物工程

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

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

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

微 Rna 对原生脂蛋白颗粒的富集及其与绝对相对微 Rna 含量及细胞转移速率的测定

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

Isolation and Analysis of Plasma Lipoproteins by Ultracentrifugation

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

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

超中心对等离子脂蛋白的分离和分析

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

Isolation and Profiling of MicroRNA-containing Exosomes from Human Bile

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

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

分离和剖析人力胆汁含外来体的MicroRNA-

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

医学

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

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

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

外来体的纯化和microRNA剖析来源于血液和文化传媒

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

生物学

Isolation of Small Noncoding RNAs from Human Serum

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

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

小非编码RNA从人血清中分离

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

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

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

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

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 ...

Rapid Lipid Droplet Isolation Protocol Using a Well-established Organelle Isolation Kit

Lipid droplets (LDs) are bioactive organelles found within the cytosol of the most eukaryotic and some prokaryotic cells. LDs are composed of neutral lipids encased by a monolayer of phospholipids and proteins. Hepatic LD lipids, such as ceramides, and proteins are implicated in several diseases that cause hepatic steatosis. Although previous methods have been established for LD isolation, they require a time-consuming preparation of reagents and are not designed for the isolation of multiple subcellular compartments. We sought to establish a new protocol to enable the isolation of LDs, endoplasmic reticulum (ER), and lysosomes from a single mouse liver.
Further, all reagents used in the protocol presented here are commercially available and require minimal reagent preparation without sacrificing LD purity. Here we present data comparing this new protocol to a standard sucrose gradient protocol, demonstrating comparable purity, morphology, and yield. Additionally, we can isolate ER and lysosomes using the same sample, providing detailed insight into the formation and intracellular flux of lipids and their associated proteins.

This protocol establishes a novel method of lipid droplet isolation and purification from mouse livers, using a well-established endoplasmic reticulum isolation kit.

利用成熟的细胞隔离试剂盒快速分离脂滴器

Lipid droplets (LDs) are bioactive organelles found within the cytosol of the most eukaryotic and some prokaryotic cells. LDs are composed of neutral ...

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

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

循环 miRNA 提取:一种通过有机提取和小 RNA 富集从血浆样品中分离 miRNA 的方法

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

从高纯高密度脂蛋白的miRNA的高通量快速分离和简化方法

摘要

探索更多视频

此视频中的章节

高密度脂蛋白的分离的非编码小RNA定量

高通量微RNA分析：优化复纳升秤定量RT - PCR Fluidigm公司动态ArrayTM国际金融中心

微 Rna 对原生脂蛋白颗粒的富集及其与绝对相对微 Rna 含量及细胞转移速率的测定

超中心对等离子脂蛋白的分离和分析

分离和剖析人力胆汁含外来体的MicroRNA-

外来体的纯化和microRNA剖析来源于血液和文化传媒

小非编码RNA从人血清中分离

小RNA分子的高效结扎的微小RNA定量高通量测序

利用成熟的细胞隔离试剂盒快速分离脂滴器

循环 miRNA 提取:一种通过有机提取和小 RNA 富集从血浆样品中分离 miRNA 的方法

从高纯高密度脂蛋白的miRNA的高通量快速分离和简化方法

摘要

探索更多视频

此视频中的章节

高密度脂蛋白的分离的非编码小RNA定量

高通量微RNA分析：优化复纳升秤定量RT - PCR Fluidig​​m公司动态ArrayTM国际金融中心

微 Rna 对原生脂蛋白颗粒的富集及其与绝对相对微 Rna 含量及细胞转移速率的测定

超中心对等离子脂蛋白的分离和分析

分离和剖析人力胆汁含外来体的MicroRNA-

外来体的纯化和microRNA剖析来源于血液和文化传媒

小非编码RNA从人血清中分离

小RNA分子的高效结扎的微小RNA定量高通量测序

利用成熟的细胞隔离试剂盒快速分离脂滴器

循环 miRNA 提取:一种通过有机提取和小 RNA 富集从血浆样品中分离 miRNA 的方法

高通量微RNA分析：优化复纳升秤定量RT - PCR Fluidigm公司动态ArrayTM国际金融中心