Name: isolation of high-density lipoproteins for non-coding small rna quantification
Uploaded: 2016-11-28T12:30:00.000+00:00
Duration: 10 min 39 s
Description: Watch this Scientific Journal Video about Isolation of High-density Lipoproteins for Non-coding Small RNA Quantification at JoVE.com

This work was supported by awards from the National Institutes of Health, National Heart, Lung and Blood Institute to K.C.V. HL128996, HL113039, and HL116263. This work was also supported by awards from the American Heart Association to K.C.V. CSA2066001, D.L.M POST26630003, and R.M.A. POST25710170.

1. HDL净化（〜5.5天） <ol><li>密度梯度离心（DGUC，〜5天） <ol><li>添加90微升的100倍的抗氧化剂，以9毫升血浆从新鲜静脉血分离。 </li><li>通过从步骤1.1.1（0.0278克/毫升溴化钾血浆）中加入0.251克溴化钾9mL的血浆从1.006克/ mL的调整加KBr等离子体密度1.025克/毫升。岩石等离子体直到所有的盐溶解在室温和转移到超速离心管，并确保所有的气泡上升到顶部。 </li><li>弯曲18-G针的针尖从前端90°1厘米，斜面朝上。用注射器和18号针头，小心地将3毫升的覆盖溶液＃1中的等离子体（ 表1）以上。 注:弯曲针可确保所有的VLDL / IDL，LDL和HDL是不与覆盖混合除去。 </li><li>去除大部分在顶部的气泡，留下2 - 从液面3毫米的空间，以在管和carefu的顶LLY置于SW-40Ti桶管。 </li><li>称重天平上每个桶（与帽），并与相对的桶（桶+帽）恰好平衡:＃1至＃4，＃2〜＃5，＃3至＃6。 注意:这些桶必须是平衡的，在任何时候都匹配。 </li><li>将转子超速（材料清单）。离心274,400 XG 24小时在4℃有4号中间制动。 </li><li>小心地从桶去除转子桶和管道。 </li><li>小心地从使用注射器和弯针的覆盖层的顶层除去2毫升。这将是可以储存于4的VLDL / IDL分数 °C或-80 C。 </li><li>去除VLDL / IDL分数后，收集剩余的7毫升样本（血浆），并把它放到一个15 mL锥形管。把样品的体积可达9毫升叠加解决方案＃2（ 表1）。 </li><li>调整从1.025克/ mL的样品密度1.080克/毫升溴化钾使用约0.746克溴化钾9毫升样品或0.0828克在/样品毫升溴化钾。直到所有的溴化钾溶解（约20分钟），并转移到超速离心管，同时确保气泡上升到顶部轻轻摇动样品。 </li><li>随着注射器和针头，小心地将3毫升的叠加解决方案＃3比样品（ 表1）。离开2 - 从液面3毫米的空间，以该管的顶部。 </li><li>在管的顶部除去大部分任何残留的气泡，并小心地在SW-40Ti桶填充超离心管中。 </li><li>称重天平上每个桶（与帽），并与相对的桶+帽恰好平衡，如在1.1.5。 </li><li>放入超速离心机转子（见材料清单）。离心274,400 XG 24小时在4℃有4号中间制动。 </li><li>小心地从桶去除转子桶和管道。 </li><li>用注射器从覆盖的顶层小心地取出2毫升，并NT针。这将是可以储存于4 LDL部分 °C或-80 C。 </li><li>去除LDL部分后，收集剩余的大约7毫升样本（血浆），并放置到一个新的15 mL锥形管。使样品（血浆）的多达9毫升的体积与1.080克/ mL溶液＃4（ 表1）。 </li><li>从1.080克/毫升调整采样密度1.30克/毫升的9毫升样本（血浆）KBr压3.33克KBr压或0.37克溴化钾样品的每毫升。岩石或稍微搅动样品，直到所有的溴化钾（盐）溶解并转移到超速离心管。 </li><li>用注射器和针头，小心地将3毫升的覆盖溶液＃5在样品（ 见表1）。 </li><li>在管的顶部除去大部分任何残留的气泡，而使从弯月2-3毫米的空间，以该管的顶部并小心地将在SW-40Ti桶填充超离心管中。 </li><li>称量每个桶（与天平上的帽），并准确平衡与对方斗+帽，如1.1.5。 </li><li>放入超速离心机转子（见材料清单）。离心274,400 XG 48小时在4℃有4号制动器。 </li><li>小心地从桶去除转子桶和管道。 </li><li>小心地从使用注射器和弯针的覆盖层的顶层除去约2mL。这是高密度脂蛋白馏分，它可以储存在4℃或-80℃。 注:按照DGUC的HDL可透析以除去高盐溶液。 </li><li>至透析，将所收集的HDL馏分在密封透析套筒（10,000 MW截止），并在1升1×PBS中透析过夜。变更透析缓冲液（1×PBS）中超过24小时的3倍。用磁力搅拌棒的PBS温柔扰动将提高透析。 </li><li>确定使用BCA方法8 DGUC-HDL的蛋白质浓度。 </li></ol></li><li> 快速蛋白质液相CHROmatography（FPLC）或尺寸排阻色谱（〜6小时） <ol><li>过滤1.2毫克在500微升DGUC-HDL（总蛋白）的通过0.22μm微离心过滤器（见材料清单），紧接在注射之前。 注:通过之前的0.22微米过滤器0.45μm的微离心过滤的DGUC-HDL样品的附加滤波可能需要一些样品。 </li><li>收集过滤DGUC-HDL样品，加载FPLC（填充）注射器，并确保有在注射器无气泡注入FPLC之前。 </li><li>用在2.6兆帕的压力下限值设定FPLC流速0.3毫升/分钟。平衡柱用缓冲液的0.2倍柱体积（大约15毫升），向样品注射之前。注射用3毫升缓冲到FPLC（注射循环）仪器样品并开始运行。收集1.5毫升的级分，共72级分。 </li></ol></li></ol> 2.高通量小分子RNA测序（sRNAseq，〜9日） <ol><li> RNA分离（约1天） <ol><li>通过使用根据制造商的说明比色试剂盒测定总胆固醇水平识别对应于DGUC-HDL的FPLC级分。使用这种FPLC设置，期望找到6 - 含DGUC-HDL 7 FPLC级分。 </li><li>收集所有从DGUC-HDL FPLC级分的体积（大约8 -1 0毫升1.45毫升馏分池卷），用浓缩10kDa的截留分子量离心过滤装置在4000 xg离心1小时，在4℃或直至HDL浓缩物大约为100微升。 </li><li>收集的HDL浓缩物，并使用BCA法8量化HDL总蛋白质浓度。 </li><li>确定HDL总蛋白的浓度最高，达到1毫克，可以从组中的每个样本被等分。例如，从每个样品的相同量的高密度脂蛋白总蛋白的分离RNA。 </li><li>使用执行RNA分离修改后的协议 （见材料清单）。 <ol><li>加10倍体积的苯酚的裂解试剂（参见材料的列表）样品和涡旋的1分钟。 </li><li>在室温下孵育5分钟。 </li><li>加氯仿（步骤2.1.5.1用酚裂解液体积的20％），剧烈摇晃15秒。 </li><li>在室温下孵育2 - 3分钟。 </li><li>离心机以12,000 xg离心在冷冻离心机15分钟，4℃。 </li><li>上层水相转移到新的管中，避免了相间。 </li><li> 100％的乙醇的1.5倍体积添加到水相，涡旋1分钟。 </li><li>商店样品至少1小时，或在-80℃过夜。 </li><li>与使用提供迷你列的RNA分离协议继续。 </li><li>用30μL的无RNase H 2 O洗脱第一直接添加的 H 2 O 15的μL到玻璃料，旋以低速（2,000 XG）处理2分钟，并然后旋转以高速（最大值）为1分钟。重复此步骤与H 2 O.另外15微升</li></ol></li><li>立即着手斯尔纳生成库或储存在-80℃。 </li></ol></li><li> 库生成（〜6小时） <ol><li>准备使用的sRNA文库生成试剂盒协议的sRNA cDNA文库，按照制造商的说明用一个修改的PCR扩增，详述如下。 <ol><li>准备在包含0.5μLDNA酶/ RNA酶的游离H 2 O冰PCR mastermix，15μLPCR混合物（PML）和1微升RNA PCR引物（RP1）每个样品（包括取样误差额外的10％）。 </li><li>添加PCR的16.5微升混合到每个（基因）样本。 </li><li>分配一个索引/条码号每个样品进行多路复用，并添加1微升的PCR引物指数（以相应的数字）到样品中。 </li><li>执行使用离心一个快速旋转。 注:总成交量应该现在是每个样品30μL。 </li><li>如于表2和3中详细进行PCR扩增的循环条件。 </li></ol></li></ol></li><li> 大小选择斯尔纳库删除适配器:适配器（〜1.5小时） <ol><li>目标:采用全自动DNA大小选择按照制造商的说明以下尺寸的参数执行库的大小选择156 bp的开始:135基点，结束:177基点，范围标志:广阔。 </li></ol></li><li> DNA清理（〜0.5小时） <ol><li>清理大小选择根据制造商的说明的sRNA的cDNA。 </li><li>洗脱干净，集中斯尔纳基因与2×7μLDNA酶/无RNase H 2 O. </li></ol></li><li> 量化和评估斯尔纳cDNA文库的产量和品质（〜1小时） <ol><li>使用高灵敏度的DNA芯片上的生物分析仪评估的sRNA cDNA文库的质量和尺寸（材料清单），为每厂家专业urer的说明。 </li><li>使用高灵敏度的DNA测定（材料清单），按照制造商的说明定量个体的sRNA cDNA文库的浓度。 注:建议有一个与不同的索引的所有样本进行上，如果可能的流动池的同一车道中运行。如果需要多于一个车道，适当地混合的情况下和对照样品。 </li></ol></li><li> 高通量的sRNA测序（〜7天） <ol><li>池个别指数基于等摩尔浓度斯尔纳库。 </li><li> 2.5.2 - 屏幕上使用生物分析仪高灵敏度的DNA芯片，并确定文库DNA浓度2.5.1合并斯尔纳库质量。 </li><li>的适配器含量进行定量PCR（定量PCR），以确保适当的深度测序使用测序文库定量PCR定量试剂盒按照制造商的说明（材料清单）。 </li><li>使用单一的读取进行测序，50 BP协议达到约20的深度 - 25男读取/样品，按照制造商的说明。 </li></ol></li></ol> 3.数据分析（约1天） <ol><li>使用bcl2fastq2，按照用户手册的说明，以解复用的样品预处理的原料FASTQ文件（材料清单）。 </li><li>使用Cutadapt修剪适配器9，并删除读&lt;16个核苷酸（NTS）长，按照用户手册的说明（材料清单）。 </li><li>除去目前在斯尔纳库，包括一站式的解决方案序列污染序列（STP:CCACGTTCCCGTGG）使用removeSequenceInFastq功能NGSPERL框架和适配器结转（CTACAGTCCGACGATC），按照用户手册的说明（材料清单）。 </li><li>通过中心的定量科学（CQS）工具FastQC执行质量控制指标，按用户手册的说明（材料清单）。 </li><li>生成的"同一"非冗余名单读起来（崩溃redundaNT读取），并使用CQSTools记录的拷贝数，按照用户手册的说明材料（列表）。 </li><li>对齐读取使用Bowtie1.1.2允许1错配的人类基因组（选项:-a -m 100 --best --strata -v 1），按照用户手册的说明（材料清单）。 </li><li>执行读取校准，计数，并使用NGSPERL，按照用户手册的说明导致摘要任务。 </li><li>转换出口计数表到电子表格文件。 </li><li>一个特定的类（ 如中，miRNA）的正常化读取到总数为类（ 例如，miRNA的总数读取）和报表信号，读取数映射的miRNA（RPMM）读取。 （ 例如 ，RPMM =（MIR-X / miRNA的总＃读取）* 1,000,000）。 </li><li>输入归伯爵表作为一般单色技术到软件的低层次和高层次的差异分析按照用户手册的说明（材料清单）。 注意:目前，没有良好的特点看家基因/ SRN至于HDL-斯尔纳。尖峰在控制都可以使用，但也可以是有问题的。正火高密度脂蛋白总蛋白输入或体积建议。最重要的是，它是推荐的各种策略之一来验证测序结果以量化使用实时PCR或定量PCR的miRNA和sRNAs。 </li></ol>

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H., Tewari, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNA+profiling:+approaches+and+considerations.">MicroRNA profiling: approaches and considerations.</a> Nat Rev Genet. 13, 358-369 (2012).</li><li>Kirschner, M. B., 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=Haemolysis+during+sample+preparation+alters+microRNA+content+of+plasma.">Haemolysis during sample preparation alters microRNA content of plasma.</a> PLoS One. 6, e24145 (2011).</li><li>Kannan, M., Atreya, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+profiling+of+human+red+blood+cells+during+storage+for+52+selected+microRNAs.">Differential profiling of human red blood cells during storage for 52 selected microRNAs.</a> Transfusion. 50, 1581-1588 (2010).</li><li>Chen, S. Y., Wang, Y., Telen, M. J., Chi, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+genomic+analysis+of+erythrocyte+microRNA+expression+in+sickle+cell+diseases.">The genomic analysis of erythrocyte microRNA expression in sickle cell diseases.</a> PLoS One. 3, e2360 (2008).</li><li>Balzano, F., 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=miRNA+Stability+in+Frozen+Plasma+Samples.">miRNA Stability in Frozen Plasma Samples.</a> Molecules. 20, 19030-19040 (2015).</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>Cheung, M. C., Wolf, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+effect+of+ultracentrifugation+on+apolipoprotein+A-I-containing+lipoprotein+subpopulations.">Differential effect of ultracentrifugation on apolipoprotein A-I-containing lipoprotein subpopulations.</a> J Lipid Res. 29, 15-25 (1988).</li><li>Kunitake, S. T., Kane, J. 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The authors have nothing to disclose.

该协议被设计为通过高通量测序或实时PCR的高纯度的HDL量化的miRNA和其他sRNAs。与任何的办法，特别考虑应给予在净化HDL和RNA的过程中的每个步骤，然后量化sRNAs。这个协议设计用于开始≥2毫升血浆的项目。然而，高质量的RNA分析可以成功地与HDL使用亲和层析从人或小鼠血浆只需80μL的纯化完成;然而，更大的起点血浆容量产生使用这里介绍的方法，更好的数据。样品处理和提取方法可以大大改变的...

外的非编码小RNA（sRNAs）代表一类新的疾病生物标志物和潜在的治疗靶点，并可能促进细胞-细胞通信1。的sRNA的研究最广泛的类型是微RNA（miRNA）的其中大约长22个核苷酸，并从长前体形式和初级转录2被处理。微RNA已被证实在转录后通过蛋白质翻译和mRNA降解2的诱导的抑制调节基因表达。然而，miRNA是只是许多类型sRNAs中的一个;作为sRNAs可以从父母的tRNA（tRNA的衍生sRNAs，TDR），核小分子RNA（斯尔纳衍生sRNAs，sndRNA），核仁小分子RNA（的snoRNA衍生sRNAs，小核RNA），核糖体RNA被切割（rRNA的衍生sRNAs，RDR ）中，Y的RNA（YDR）和其他杂项的RNA 1。这些新颖sRNAs的几个例子已经被报道功能类似的miRNA;然而，生物福许多这些sRNAs的nctions仍有待确定，尽管在基因调控作用可能3-6。最有趣的是，微RNA等sRNAs是在细胞外流体，包括唾液，血浆，尿和胆汁稳定地存在。胞sRNAs有可能通过其与细胞外囊泡（EV），脂蛋白，和/或细胞外核糖核蛋白复合物关联免受RNA酶。 先前，我们报道了脂蛋白，即高密度脂蛋白（HDL），在等离子体7运输的miRNA。在这项研究中，高密度脂蛋白是使用密度梯度超速离心（DGUC），快速蛋白液相色谱法（尺寸排阻色谱凝胶过滤，FPLC），顺序法和亲和层析（抗载脂蛋白AI（载脂蛋白A-I）的免疫沉淀分离）7。同时使用基于PCR的实时低密度阵列和单个miRNA的测定法，miRNA的水平对HDL定量从愈合分离你和高胆固醇血症患者7。使用这种方法，我们可以来分析miRNAs与量化高纯HDL准备具体的miRNA。自2011年以来，我们已经确定，尽管亲和层析提高HDL纯度，饱和度抗体大大限制了产量，并且可以成本过高。目前，我们的协议建议随后通过FPLC，也产生高品质的HDL样品下游RNA分离和的sRNA量化DGUC的两步骤顺序串联方式。由于sRNAs（sRNAseq）， 例如 ，miRNA的高通量测序，并增加了其他非miRNA的斯尔纳类的认识的最新进展，sRNAseq是国家的最先进的miRNA与斯尔纳分析的电流。因此，我们的协议建议量化miRNAs与使用sRNAseq HDL样品等sRNAs。尽管如此，从HDL中分离总RNA也可用于量化个体的miRNA和其他sRNAs或使用实时PCR一验证sRNAseq结果pproaches。在这里，我们详细描述的收集，纯化，量化，数据分析和高纯度的高密度脂蛋白sRNAs的验证协议。 本文的总体目标是展示从人血浆中分离的高纯度的HDL的sRNA量化的可行性和工艺。

<table><tbody><tr><td>Ultracentrifuge&amp;nbsp;</td><td>Beckman Coulter</td><td>A99839</td><td>Optima XPN-80</td></tr><tr><td>Ultracentrifuge Rotor</td><td>Beckman Coulter</td><td>331362</td><td>SW-41Ti</td></tr><tr><td>AKTA Pure FPLC System</td><td>GE Healthcare</td><td>29018224</td><td></td></tr><tr><td>3x FPLC Superdex 200 Increase Columns In-line</td><td>GE Healthcare</td><td>28990944</td><td>10/300 gl</td></tr><tr><td>SynergyMx</td><td>BioTek Instruments</td><td>7191000</td><td></td></tr><tr><td>Tabletop centrifuge</td><td>Thermo Scientific</td><td>75004525</td><td>Sorvall ST40R</td></tr><tr><td>Refrigerated centrifuge</td><td>Eppendorf</td><td>22629867</td><td>5417R (purchased through USA Scientific)</td></tr><tr><td>Microfuge&amp;nbsp;</td><td>USA Scientific</td><td>2631-0006</td><td></td></tr><tr><td>PippenPrep</td><td>Sage Science</td><td>PIP0001</td><td></td></tr><tr><td>2100 Bioanalyzer&amp;nbsp;</td><td>Agilent</td><td>G2938B</td><td></td></tr><tr><td>High Sensitivity DNA Assay</td><td>Agilent</td><td>5067-4626</td><td></td></tr><tr><td>Sequencing Library qPCR Quantification Kit</td><td>Illumina</td><td>SY-930-1010</td><td></td></tr><tr><td>ProFlex Thermal Cycler</td><td>Applied Biosystems</td><td>4484073</td><td></td></tr><tr><td>QuantStudio 12k Flex</td><td>Applied Biosystems</td><td>4471134</td><td></td></tr><tr><td>EpMotion Robot</td><td>Eppendorf</td><td>960000111</td><td>5070</td></tr><tr><td>Ultra-clear centrifuge tubes</td><td>Beckman Coulter</td><td>344059</td><td></td></tr><tr><td>Potassium Bromide</td><td>Fisher Chemicals</td><td>P205-500</td><td></td></tr><tr><td>15 mL conical tube</td><td>Thermo Scientific</td><td>339650</td><td></td></tr><tr><td>Micro-centrifugal filters 0.45 &amp;micro;m</td><td>Millipore</td><td>UFC30HV00</td><td></td></tr><tr><td>Micro-centrifugal filters 0.22 &amp;micro;m</td><td>Millipore</td><td>UFC30GV00</td><td></td></tr><tr><td>miRNAEasy Total RNA Isolation Kits</td><td>Qiagen</td><td>217004</td><td></td></tr><tr><td>Total Cholesterol colormetric kit</td><td>Cliniqa (Raichem)</td><td>R80035</td><td></td></tr><tr><td>10,000 m.w. cut-off centrifugation filter</td><td>Amicon</td><td>UFC801024</td><td>purchased through Millipore</td></tr><tr><td>PCR strip tubes</td><td>Axygen</td><td>PCR-0208-C</td><td>purchased through Fisher</td></tr><tr><td>microRNA RT kit</td><td>Life Technologies</td><td>4366597</td><td>For 1000 reactions</td></tr><tr><td>PCR master mix</td><td>Life Technologies</td><td>4440041</td><td>50 mL bottle</td></tr><tr><td>Pierce BCA kit</td><td>Thermo Scientific</td><td>23225</td><td></td></tr><tr><td>Clean and Concentrator Kit</td><td>Zymo</td><td>D4014</td><td></td></tr><tr><td>Dialysis tubing</td><td>Spectrum Labs</td><td>132118</td><td>purchased through Fisher</td></tr><tr><td>bcl2fastq2</td><td>Illumina</td><td>n/a</td><td>Software</td></tr><tr><td>Cutadapt</td><td>https://github.com/marcelm/cutadapt</td><td>n/a</td><td>Software</td></tr><tr><td>NGSPERL</td><td>github.com/shengqh/ngsperl</td><td>n/a</td><td>Software</td></tr><tr><td>CQSTools</td><td>github.com/shengqh/CQS.Tools</td><td>n/a</td><td>Software</td></tr><tr><td>Bowtie 1.1.2&amp;nbsp;</td><td>http://bowtie-bio.sourceforge.net</td><td>n/a</td><td>Software</td></tr><tr><td>GeneSpringGX13.1.1</td><td>Agilent</td><td>n/a</td><td>Software</td></tr></tbody></table>

isolation of high-density lipoproteins for non-coding small rna quantification

的小非编码RNA的多样性（的sRNA）正在迅速扩大以及它们在生物过程，包括基因调控作用，正在出现。最有趣的是，sRNAs也发现的细胞外，并稳定地存在于所有生物流体。因此，胞外sRNAs代表一类新的疾病的生物标志物，并有可能涉及细胞信号传导和细胞间通讯网络。以评估其作为生物标志物的潜力，sRNAs可在血浆，尿和其他流体来量化。然而，为了完全理解外sRNAs内分泌信号的影响，重要的是，以确定哪些载波被运送并保护它们在生物流体（ 例如，血浆），该细胞和组织向细胞外的sRNA池，以及细胞和能够组织接受和利用外斯尔纳。为了实现这些目标，它是分离外载体的高纯度的种群临界对的sRNA分析和定量。我们以前表明，脂蛋白，特别是高密度脂蛋白（HDL），输送单元和高密度脂蛋白的miRNA之间的功能的微RNA（miRNA）的疾病是显著改变。这里，我们详细，利用与密度梯度超速离心法（DGUC）和快速蛋白液相层析（FPLC）串联的HDL隔离一个新的协议，以获得高纯度的HDL用于下游分析和所有sRNAs，包括miRNA的量化，同时使用高-throughput测序和实时PCR的方法。该协议将是对HDL sRNAs调查的宝贵资源。

这个协议是一系列连接在一起，以允许sRNAs对高纯度的HDL通过高通量测序或实时PCR（ 图1）的定量确定的方法。为了演示的可行性和这个协议的影响，高密度脂蛋白是从人血浆中由串联DGUC和FPLC方法纯化。对应于高密度脂蛋白（由胆固醇分布）收集FPLC级分浓缩和总RNA，1毫克的HDL（总蛋白）的分离。从N = 4的健康人体内高密度脂蛋白收集生成斯尔纳库。人类所有?...

Watch this Scientific Journal Video about Isolation of High-density Lipoproteins for Non-coding Small RNA Quantification at JoVE.com

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

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 ...

isolation-high-density-lipoproteins-for-non-coding-small-rna

Research

JoVE Journal

Biochemistry

11.3K 次观看.  Vanderbilt University School of Medicine. This protocol describes the isolation and quantification of high-density lipoprotein small RNAs.

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

Isolation of Cognate RNA-protein Complexes from Cells Using Oligonucleotide-directed Elution

Ribonucleoprotein particles direct the biogenesis and post-transcriptional regulation of all mRNAs through distinct combinations of RNA binding proteins. They are composed of position-dependent, cis-acting RNA elements and unique combinations of RNA binding proteins. Defining the composition of a specific RNP is essential to achieving a fundamental understanding of gene regulation. The isolation of a select RNP is akin to finding a needle in a haystack. Here, we demonstrate an approach to isolate RNPs associated at the 5' untranslated region of a select mRNA in asynchronous, transfected cells. This cognate RNP has been demonstrated to be necessary for the translation of select viruses and cellular stress-response genes.
The demonstrated RNA-protein co-precipitation protocol is suitable for the downstream analysis of protein components through proteomic analyses, immunoblots, or suitable biochemical identification assays. This experimental protocol demonstrates that DHX9/RNA helicase A is enriched at the 5' terminus of cognate retroviral RNA and provides preliminary information for the identification of its association with cell stress-associated huR and junD cognate mRNAs.

This manuscript describes an approach to isolate select cognate RNPs formed in eukaryotic cells via a specific oligonucleotide-directed enrichment. We demonstrate the applicability of this approach by isolating a cognate RNP bound to the retroviral 5' untranslated region that is composed of DHX9/RNA helicase A.

从细胞使用寡核苷酸定向洗脱同源RNA-蛋白质复合物的隔离

Ribonucleoprotein particles direct the biogenesis and post-transcriptional regulation of all mRNAs through distinct combinations of RNA binding proteins. ...

科研

生物化学

Lipid Droplet Isolation for Quantitative Mass Spectrometry Analysis

Lipid droplets are vital to the replication of a variety of different pathogens, most prominently the Hepatitis C Virus (HCV), as the putative site of virion morphogenesis. Quantitative lipid droplet proteome analysis can be used to identify proteins that localize to or are displaced from lipid droplets under conditions such as virus infections. Here, we describe a protocol that has been successfully used to characterize the changes in the lipid droplet proteome following infection with HCV. We use Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC) and thus label the complete proteome of one population of cells with &#34;heavy&#34; amino acids to quantitate the proteins by mass spectrometry. For lipid droplet isolation, the two cell populations (i.e.&#160;HCV-infected/&#34;light&#34; amino acids and uninfected control/&#34;heavy&#34; amino acids) are mixed 1:1 and lysed mechanically in hypotonic buffer. After removing the nuclei and cell debris by low speed centrifugation, lipid droplet-associated proteins are enriched by two subsequent ultracentrifugation steps followed by three washing steps in isotonic buffer. The purity of the lipid droplet fractions is analyzed by western blotting with antibodies recognizing different subcellular compartments. Lipid droplet-associated proteins are then separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) followed by Coomassie staining. After tryptic digest, the peptides are quantified by liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS). Using this method, we identified proteins recruited to lipid droplets upon HCV infection that might represent pro- or antiviral host factors. Our method can be applied to a variety of different cells and culture conditions, such as infection with pathogens, environmental stress, or drug treatment.

Lipid droplets are important organelles for the replication of several pathogens, including the Hepatitis C Virus (HCV). We describe a method to isolate lipid droplets for quantitative mass spectrometry of associated proteins; it can be used under a variety of conditions, such as virus infection, environmental stress, or drug treatment.

脂滴隔离定量质谱分析

Lipid droplets are vital to the replication of a variety of different pathogens, most prominently the Hepatitis C Virus (HCV), as the putative site of ...

A Complete Pipeline for Isolating and Sequencing MicroRNAs, and Analyzing Them Using Open Source Tools

Half of all human transcripts are thought to be regulated by microRNAs. Therefore, quantifying microRNA expression can reveal underlying mechanisms in disease states and provide therapeutic targets and biomarkers. Here, we detail how to accurately quantify microRNAs. Briefly, this method describes isolating microRNAs, ligating them to adaptors suitable for high-throughput sequencing, amplifying the final products, and preparing a sample library. Then, we explain how to align the obtained sequencing reads to microRNA hairpins, and quantify, normalize, and calculate their differential expression. Versatile and robust, this combined experimental workflow and bioinformatic analysis enables users to begin with tissue extraction and finish with microRNA quantification.

Here, we describe a step-by-step strategy for isolating small RNAs, enriching for microRNAs, and preparing samples for high-throughput sequencing. We then describe how to process sequence reads and align them to microRNAs, using open source tools.

用于隔离和测序微RNA并使用开源工具分析微RNA的完整管道

Half of all human transcripts are thought to be regulated by microRNAs. Therefore, quantifying microRNA expression can reveal underlying mechanisms in ...

遗传学

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 ...

Genome-wide Analysis of HDAC Inhibitor-mediated Modulation of microRNAs and mRNAs in B Cells Induced to Undergo Class-switch DNA Recombination and Plasma Cell Differentiation

Antibody responses are accomplished through several critical B cell-intrinsic processes, including somatic hypermutation (SHM), class-switch DNA recombination (CSR), and plasma cell differentiation. In recent years, epigenetic modifications or factors, such as histone deacetylation and microRNAs (miRNAs), have been shown to interact with B-cell genetic programs to shape antibody responses, while the dysfunction of epigenetic factors has been found to lead to autoantibody responses. Analyzing genome-wide miRNA and mRNA expression in B cells in response to epigenetic modulators is important for understanding the epigenetic regulation of B-cell function and antibody response. Here, we demonstrate a protocol for inducing B cells to undergo CSR and plasma cell differentiation, treating these B cells with histone deacetylase (HDAC) inhibitors (HDIs), and analyzing mRNA and microRNA expression. In this protocol, we directly analyze complementary DNA (cDNA) sequences using next-generation mRNA sequencing (mRNA-seq) and miRNA-seq technologies, mapping of the sequencing reads to the genome, and quantitative reverse transcription (qRT)-PCR. With these approaches, we have defined that, in B cells induced to undergo CSR and plasma cell differentiation, HDI, an epigenetic regulator, selectively modulates miRNA and mRNA expression and alters CSR and plasma cell differentiation.

Epigenetic factors can interact with genetic programs to modulate gene expression and regulate B cell function. By combining in vitro B-cell stimulation, qRT-PCR, and high-throughput microRNA-sequence and mRNA-sequence approaches, we can analyze the epigenetic modulation of miRNA and gene expression in B cells.

HDAC 抑制剂介导的 B 细胞 rna 和基因的基因组分析--诱导进行类开关 DNA 重组和血浆细胞分化

Antibody responses are accomplished through several critical B cell-intrinsic processes, including somatic hypermutation (SHM), class-switch DNA ...

免疫与感染

Isolation and Characterization of RNA-Containing Exosomes

The field of exosome research is rapidly expanding, with a dramatic increase in publications in recent years. These small vesicles (30-100 nm) of endocytic origin were first proposed to function as a way for reticulocytes to eradicate the transferrin receptor while maturing into erythrocytes1, and were later named exosomes. Exosomes are formed by inward budding of late endosomes, producing multivesicular bodies (MVBs), and are released into the environment by fusion of the MVBs with the plasma membrane2. Since the first discovery of exosomes, a wide range of cells have been shown to release these vesicles. Exosomes have also been detected in several biological fluids, including plasma, nasal lavage fluid, saliva and breast milk3-6. Furthermore, it has been demonstrated that the content and function of exosomes depends on the originating cell and the conditions under which they are produced. A variety of functions have been demonstrated for exosomes, such as induction of tolerance against allergen7,8, eradication of established tumors in mice9, inhibition and activation of natural killer cells10-12, promotion of differentiation into T regulatory cells13, stimulation of T cell proliferation14 and induction of T cell apoptosis15. Year 2007 we demonstrated that exosomes released from mast cells contain messenger RNA (mRNA) and microRNA (miRNA), and that the RNA can be shuttled from one cell to another via exosomes. In the recipient cells, the mRNA shuttled by exosomes was shown to be translated into protein, suggesting a regulatory function of the transferred RNA16. Further, we have also shown that exosomes derived from cells grown under oxidative stress can induce tolerance against further stress in recipient cells and thus suggest a biological function of the exosomal shuttle RNA17. Cell culture media and biological fluids contain a mixture of vesicles and shed fragments. A high quality isolation method for exosomes, followed by characterization and identification of the exosomes and their content, is therefore crucial to distinguish exosomes from other vesicles and particles. Here, we present a method for the isolation of exosomes from both cell culture medium and body fluids. This isolation method is based on repeated centrifugation and filtration steps, followed by a final ultracentrifugation step in which the exosomes are pelleted. Important methods to identify the exosomes and characterize the exosomal morphology and protein content are highlighted, including electron microscopy, flow cytometry and Western blot. The purification of the total exosomal RNA is based on spin column chromatography and the exosomal RNA yield and size distribution is analyzed using a Bioanalyzer.

This paper demonstrates methods for the isolation, purification and detection of exosomes, as well as techniques for analysis of their molecular content. These methods are adaptable for exosome isolation from both cell culture media and biological fluids, and can beyond analysis of molecular content also be useful in functional studies.

RNA含外来体的分离和鉴定

The field of exosome research is rapidly expanding, with a dramatic increase in publications in recent years. These small vesicles (30-100 nm) of ...

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 ...

Isolation of Viable Adipocytes and Stromal Vascular Fraction from Human Visceral Adipose Tissue Suitable for RNA Analysis and Macrophage Phenotyping

Visceral adipose tissue (VAT) is an active metabolic organ composed mainly of mature adipocytes and stromal vascular fraction (SVF) cells, which release different bioactive molecules that control metabolic, hormonal, and immune processes; currently, it is unclear how these processes are regulated within the adipose tissue. Therefore, the development of methods evaluating the contribution of each cell population to the pathophysiology of adipose tissue is crucial. This protocol describes the isolation steps and provides the necessary troubleshooting guidelines for efficient isolation of viable mature adipocytes and SVF from human VAT biopsies in a single process, using a collagenase enzymatic digestion technique. Moreover, the protocol is also optimized to identify macrophage subsets and perform mature adipocyte RNA isolation for gene expression studies, which allows performing studies dissecting the interaction between these cell populations. Briefly, VAT biopsies are washed, minced mechanically, and digested to generate a single-cell suspension. After centrifugation, mature adipocytes are isolated by flotation from the SVF pellet. The RNA extraction protocol ensures a high yield of total RNA (including miRNAs) from adipocytes for downstream expression assays. Simultaneously, SVF cells are used to characterize macrophage subsets (pro- and anti-inflammatory phenotype) through flow cytometry analysis.

This protocol provides an efficient collagenase digestion method for isolation of viable adipocytes and stromal vascular fraction-SVF cells from human visceral fat in a single process, including methodology to obtain high-quality RNA from adipocytes and phenotypification of SVF-macrophages through staining of multiple membrane-bound markers for analysis by flow cytometry.

从人内脏脂肪组织中分离出活的脂肪细胞和基质血管组分，适用于 RNA 分析和巨噬细胞表型分析

Visceral adipose tissue (VAT) is an active metabolic organ composed mainly of mature adipocytes and stromal vascular fraction (SVF) cells, which release ...

医学

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.

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.

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 ...