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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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. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Factors+affecting+the+integrity+of+high+density+lipoproteins+in+the+ultracentrifuge.">Factors affecting the integrity of high density lipoproteins in the ultracentrifuge.</a> J Lipid Res. 23, 936-940 (1982).</li><li>Laurent, L. 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=Meeting+report:+discussions+and+preliminary+findings+on+extracellular+RNA+measurement+methods+from+laboratories+in+the+NIH+Extracellular+RNA+Communication+Consortium.">Meeting report: discussions and preliminary findings on extracellular RNA measurement methods from laboratories in the NIH Extracellular RNA Communication Consortium.</a> J Extracell Vesicles. 4, 26533 (2015).</li></ol>

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 border="0" cellpadding="0" cellspacing="0" style="width:1033px;" width="1033">
	<tbody>
		<tr height="21">
			<td height="21" style="width:315px;height:21px;">Ultracentrifuge&#160;</td>
			<td style="width:207px;">Beckman Coulter</td>
			<td style="width:205px;">A99839</td>
			<td style="width:307px;">Optima XPN-80</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:315px;height:21px;">Ultracentrifuge Rotor</td>
			<td style="width:207px;">Beckman Coulter</td>
			<td style="width:205px;">331362</td>
			<td>SW-41Ti</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:315px;height:21px;">AKTA Pure FPLC System</td>
			<td style="width:207px;">GE Healthcare</td>
			<td style="width:205px;">29018224</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:315px;height:21px;">3X FPLC Superdex 200 Increase Columns In-line</td>
			<td style="width:207px;">GE Healthcare</td>
			<td style="width:205px;">28990944</td>
			<td>10/300 gl</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:315px;height:21px;">SynergyMx</td>
			<td style="width:207px;">BioTek Instruments</td>
			<td style="width:205px;">7191000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:315px;height:21px;">Tabletop centrifuge</td>
			<td style="width:207px;">Thermo Scientific</td>
			<td style="width:205px;">75004525</td>
			<td>Sorvall ST40R</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:315px;height:21px;">Refrigerated centrifuge</td>
			<td style="width:207px;">Eppendorf</td>
			<td style="width:205px;">22629867</td>
			<td>5417R (purchased through USA Scientific)</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:315px;height:21px;">Microfuge&#160;</td>
			<td style="width:207px;">USA Scientific</td>
			<td style="width:205px;">2631-0006</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:315px;height:21px;">PippenPrep</td>
			<td style="width:207px;">Sage Science</td>
			<td style="width:205px;">PIP0001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:315px;height:21px;">2100 Bioanalyzer&#160;</td>
			<td style="width:207px;">Agilent</td>
			<td>G2938B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:315px;height:21px;">High Sensitivity DNA Assay</td>
			<td style="width:207px;">Agilent</td>
			<td>5067-4626</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:315px;height:21px;">Sequencing Library qPCR Quantification Kit</td>
			<td style="width:207px;">Illumina</td>
			<td>SY-930-1010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:315px;height:21px;">ProFlex Thermal Cycler</td>
			<td style="width:207px;">Applied Biosystems</td>
			<td style="width:205px;">4484073</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:315px;height:21px;">QuantStudio 12k Flex</td>
			<td style="width:207px;">Applied Biosystems</td>
			<td style="width:205px;">4471134</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:315px;height:21px;">EpMotion Robot</td>
			<td style="width:207px;">Eppendorf</td>
			<td style="width:205px;">960000111</td>
			<td>5070</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:315px;height:21px;">Ultra-clear centrifuge tubes</td>
			<td style="width:207px;">Beckman Coulter</td>
			<td style="width:205px;">344059</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:315px;height:21px;">Potassium Bromide</td>
			<td style="width:207px;">Fisher Chemicals</td>
			<td style="width:205px;">P205-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:315px;height:21px;">15 mL conical tube</td>
			<td style="width:207px;">Thermo Scientific</td>
			<td style="width:205px;">339650</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:315px;height:21px;">Micro-centrifugal filters 0.45&#181;m</td>
			<td style="width:207px;">Millipore</td>
			<td style="width:205px;">UFC30HV00</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:315px;height:21px;">Micro-centrifugal filters 0.22&#181;m</td>
			<td style="width:207px;">Millipore</td>
			<td style="width:205px;">UFC30GV00</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:315px;height:21px;">miRNAEasy Total RNA Isolation Kits</td>
			<td style="width:207px;">Qiagen</td>
			<td style="width:205px;">217004</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:315px;height:21px;">Total Cholesterol colormetric kit</td>
			<td style="width:207px;">Cliniqa (Raichem)</td>
			<td style="width:205px;">R80035</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:315px;height:21px;">10,000 m.w. cut-off centrifugation filter</td>
			<td style="width:207px;">Amicon</td>
			<td style="width:205px;">UFC801024</td>
			<td>purchased through Millipore</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:315px;height:21px;">PCR strip tubes</td>
			<td style="width:207px;">Axygen</td>
			<td style="width:205px;">PCR-0208-C</td>
			<td>purchased through Fisher</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:315px;height:21px;">microRNA RT kit</td>
			<td style="width:207px;">Life Technologies</td>
			<td style="width:205px;">4366597</td>
			<td>For 1000 reactions</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:315px;height:21px;">PCR master mix</td>
			<td style="width:207px;">Life Technologies</td>
			<td style="width:205px;">4440041</td>
			<td>50 mL bottle</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:315px;height:21px;">Pierce BCA kit</td>
			<td style="width:207px;">Thermo Scientific</td>
			<td style="width:205px;">23225</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:315px;height:21px;">Clean and Concentrator Kit</td>
			<td style="width:207px;">Zymo</td>
			<td style="width:205px;">D4014</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:315px;height:21px;">Dialysis tubing</td>
			<td style="width:207px;">Spectrum Labs</td>
			<td style="width:205px;">132118</td>
			<td>purchased through Fisher</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:315px;height:21px;">bcl2fastq2</td>
			<td style="width:207px;">Illumina</td>
			<td style="width:205px;">n/a</td>
			<td>Software</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:315px;height:21px;">Cutadapt</td>
			<td>https://github.com/marcelm/cutadapt</td>
			<td style="width:205px;">n/a</td>
			<td>Software</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:315px;height:21px;">NGSPERL</td>
			<td>github.com/shengqh/ngsperl</td>
			<td style="width:205px;">n/a</td>
			<td>Software</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:315px;height:21px;">CQSTools</td>
			<td>github.com/shengqh/CQS.Tools</td>
			<td style="width:205px;">n/a</td>
			<td>Software</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:315px;height:21px;">Bowtie 1.1.2&#160;</td>
			<td>http://bowtie-bio.sourceforge.net</td>
			<td style="width:205px;">n/a</td>
			<td>Software</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:315px;height:21px;">GeneSpringGX13.1.1</td>
			<td style="width:207px;">Agilent</td>
			<td style="width:205px;">n/a</td>
			<td>Software</td>
		</tr>
	</tbody>
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
</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

11.2K 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

An Efficient Method for the Isolation of Highly Purified RNA from Seeds for Use in Quantitative Transcriptome Analysis

Plant seeds accumulate large amounts of storage reserves comprising biodegradable organic matter. Humans rely on seed storage reserves for food and as industrial materials. Gene expression profiles are powerful tools for investigating metabolic regulation in plant cells. Therefore, detailed, accurate gene expression profiles during seed development are required for crop breeding. Acquiring highly purified RNA is essential for producing these profiles. Efficient methods are needed to isolate highly purified RNA from seeds. Here, we describe a method for isolating RNA from seeds containing large amounts of oils, proteins, and polyphenols, which have inhibitory effects on high-purity RNA isolation. Our method enables highly purified RNA to be obtained from seeds without the use of phenol, chloroform, or additional processes for RNA purification. This method is applicable to Arabidopsis, rapeseed, and soybean seeds. Our method will be useful for monitoring the expression patterns of low level transcripts in developing and mature seeds.

We have succeeded in establishing a method for RNA isolation from plant seeds containing large amounts of oils, proteins, and polyphenols, which have inhibitory effects on high-purity RNA isolation. Our method is suitable for monitoring the expression of genes with low level transcripts in seeds.

对于高度纯化RNA的种子定量转录组分析的隔离中使用的有效方法

Plant seeds accumulate large amounts of storage reserves comprising biodegradable organic matter. Humans rely on seed storage reserves for food and as ...

Toeprinting Analysis of Translation Initiation Complex Formation on Mammalian mRNAs

Translation initiation is the rate-limiting step of protein synthesis and represents a key point at which cells regulate their protein output. Regulation of protein synthesis is the key to cellular stress-response, and dysregulation is central to many disease states, such as cancer. For instance, although cellular stress leads to the inhibition of global translation by attenuating cap-dependent initiation, certain stress-response proteins are selectively translated in a cap-independent manner. Discreet RNA regulatory elements, such as cellular internal ribosome entry sites (IRESes), allow for the translation of these specific mRNAs. Identification of such mRNAs, and the characterization of their regulatory mechanisms, have been a key area in molecular biology. Toeprinting is a method for the study of RNA structure and function as it pertains to translation initiation. The goal of toeprinting is to assess the ability of in vitro transcribed RNA to form stable complexes with ribosomes under a variety of conditions, in order to determine which sequences, structural elements, or accessory factors are involved in ribosome binding&#8212;a pre-cursor for efficient translation initiation. Alongside other techniques, such as western analysis and polysome profiling, toeprinting allows for a robust characterization of mechanisms for the regulation of translation initiation.

Toeprinting aims to measure the ability of in vitro transcribed RNA to form translation initiation complexes with ribosomes under a variety of conditions. This protocol describes a method for toeprinting mammalian RNA and can be used to study both cap-dependent and IRES-driven translation.

哺乳动物基因翻译起始复合体形成的 Toeprinting 分析

Translation initiation is the rate-limiting step of protein synthesis and represents a key point at which cells regulate their protein output. Regulation ...

An Oligonucleotide-based Tandem RNA Isolation Procedure to Recover Eukaryotic mRNA-Protein Complexes

RNA-binding proteins (RBPs) play key roles in the post-transcriptional control of gene expression. Therefore, biochemical characterization of mRNA-protein complexes is essential to understanding mRNA regulation inferred by interacting proteins or non-coding RNAs. Herein, we describe a tandem RNA isolation procedure (TRIP) that enables the purification of endogenously formed mRNA-protein complexes from cellular extracts. The two-step protocol involves the isolation of polyadenylated mRNAs with antisense oligo(dT) beads and subsequent capture of an mRNA of interest with 3&#39;-biotinylated 2&#39;-O-methylated antisense RNA oligonucleotides, which can then be isolated with streptavidin beads. TRIP was used to recover in vivo crosslinked mRNA-ribonucleoprotein (mRNP) complexes from yeast, nematodes and human cells for further RNA and protein analysis. Thus, TRIP is a versatile approach that can be adapted to all types of polyadenylated RNAs across organisms to study the dynamic re-arrangement of mRNPs imposed by intracellular or environmental cues.

A tandem RNA isolation procedure (TRIP) for recovery of endogenously formed mRNA-protein complexes is described. Specifically, RNA-protein complexes are crosslinked in vivo, polyadenylated RNAs are isolated from extracts with oligo(dT) beads, and particular mRNAs are captured with modified RNA antisense oligonucleotides. Proteins bound to mRNAs are detected by immunoblot analysis.

基于寡核苷酸的串联 RNA 分离法恢复真核 mRNA-蛋白复合物

RNA-binding proteins (RBPs) play key roles in the post-transcriptional control of gene expression. Therefore, biochemical characterization of mRNA-protein ...

Structural Studies of Macromolecules in Solution using Small Angle X-Ray Scattering

Protein-protein interactions involving proteins with multiple globular domains present technical challenges for determining how such complexes form and how the domains are oriented/positioned. Here, a protocol with the potential for elucidating which specific domains mediate interactions in multicomponent system through ab initio modeling is described. A method for calculating solution structures of macromolecules and their assemblies is provided that involves integrating data from small angle X-ray scattering (SAXS), chromatography, and atomic resolution structures together in a hybrid approach. A specific example is that of the complex of full-length nidogen-1, which assembles extracellular matrix proteins and forms an extended, curved nanostructure. One of its globular domains attaches to laminin &#947;-1, which structures the basement membrane. This provides a basis for determining accurate structures of flexible multidomain protein complexes and is enabled by synchrotron sources coupled with automation robotics and size exclusion chromatography systems. This combination allows rapid analysis in which multiple oligomeric states are separated just prior to SAXS data collection. The analysis yields information on the radius of gyration, particle dimension, molecular shape and interdomain pairing. The protocol for generating 3D models of complexes by fitting high-resolution structures of the component proteins is also given.

Here, we present how Small Angle X-Ray Scattering (SAXS) can be utilized to obtain information on low-resolution envelopes representing the macromolecular structures. When used in conjunction with high-resolution structural techniques such as X-Ray Crystallography and Nuclear Magnetic Resonance, SAXS can provide detailed insights into multidomain proteins and macromolecular complexes in-solution.

小角度 x 射线散射溶液中大分子的结构研究

Protein-protein interactions involving proteins with multiple globular domains present technical challenges for determining how such complexes form and ...

Capture and Identification of RNA-binding Proteins by Using Click Chemistry-assisted RNA-interactome Capture (CARIC) Strategy

A comprehensive identification of RNA-binding proteins (RBPs) is key to understanding the posttranscriptional regulatory network in cells. A widely used strategy for RBP capture exploits the polyadenylation [poly(A)] of target RNAs, which mostly occurs on eukaryotic mature mRNAs, leaving most binding proteins of non-poly(A) RNAs unidentified. Here we describe the detailed procedures of a recently reported method termed click chemistry-assisted RNA-interactome capture (CARIC), which enables the transcriptome-wide capture of both poly(A) and non-poly(A) RBPs by combining the metabolic labeling of RNAs, in vivo UV cross-linking, and bioorthogonal tagging.

Capture and Identification of RNA-binding Proteins by Using Click Chemistry-assisted RNA-interactome Capture CARIC Strategy

A detailed protocol for applying the click chemistry-assisted RNA-interactome capture (CARIC) strategy to identify proteins binding to both coding and noncoding RNAs is presented.

利用单击化学辅助 rna-路特捕获 (无花果) 策略捕获和鉴定 rna 结合蛋白

A comprehensive identification of RNA-binding proteins (RBPs) is key to understanding the posttranscriptional regulatory network in cells. A widely used ...

Studying RNA Interactors of Protein Kinase RNA-Activated during the Mammalian Cell Cycle

Protein kinase RNA-activated (PKR) is a member of the innate immune response proteins and recognizes the double-stranded secondary structure of viral RNAs. When bound to viral double-stranded RNAs (dsRNAs), PKR undergoes dimerization and subsequent autophosphorylation. Phosphorylated PKR (pPKR) becomes active and induces phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF2&#945;) to suppress global translation. Increasing evidence suggests that PKR can be activated under physiological conditions such as during the cell cycle or under various stress conditions without infection. However, our understanding of the RNA activators of PKR is limited due to the lack of a standardized experimental method to capture and analyze PKR-interacting dsRNAs. Here, we present an experimental protocol to specifically enrich and analyze PKR bound RNAs during the cell cycle using HeLa cells. We utilize the efficient crosslinking activity of formaldehyde to fix PKR-RNA complexes and isolate them via immunoprecipitation. PKR co-immunoprecipitated RNAs can then be further processed to generate a high-throughput sequencing library. One major class of PKR-interacting cellular dsRNAs is mitochondrial RNAs (mtRNAs), which can exist as intermolecular dsRNAs through complementary interaction between the heavy-strand and the light-strand RNAs. To study the strandedness of these duplex mtRNAs, we also present a protocol for strand-specific qRT-PCR. Our protocol is optimized for the analysis of PKR-bound RNAs, but it can be easily modified to study cellular dsRNAs or RNA-interactors of other dsRNA binding proteins.

We present experimental approaches for studying RNA-interactors of double-stranded RNA binding protein kinase RNA-activated (PKR) during the mammalian cell cycle using HeLa cells. This method utilizes formaldehyde to crosslink RNA-PKR complexes and immunoprecipitation to enrich PKR-bound RNAs. These RNAs can be further analyzed through high-throughput sequencing or qRT-PCR.

哺乳动物细胞周期内蛋白质激酶 rna 激活的 rna 相互作用研究

Protein kinase RNA-activated (PKR) is a member of the innate immune response proteins and recognizes the double-stranded secondary structure of viral ...

Translating Ribosome Affinity Purification (TRAP) for RNA Isolation from Endothelial Cells In Vivo

Many studies have been limited to using in vitro cellular assays and whole tissues or isolating of specific cell types from animals for in vitro analysis of transcriptome and gene expression by qPCR and RNA sequencing. Comprehensive transcriptome and gene expression analysis of specific cell types in complex tissues and organs will be critical to understand cellular and molecular mechanisms by which genes are regulated and their association with tissue homeostasis and organ functions. In this article, we demonstrate the methodology for isolation of ribosome-bound RNA directly in vivo in the vascular endothelia of animal lungs as an example. The specific materials and procedures for tissue processing and RNA purification will be described, including the assessment of RNA quality and yield as well as real time qPCR for arteriogenic gene assays. This approach, known as translating ribosome affinity purification (TRAP) technique, can be utilized for characterization of gene expression and transcriptome analysis of certain cell types directly in vivo in any specific type in complex tissues.

Translating Ribosome Affinity Purification TRAP for RNA Isolation from Endothelial Cells In Vivo

We present an approach to purify ribosome-bound mRNA from vascular endothelial cells (ECs) directly in mouse brain, lung and heart tissues via EC-specific genetic tag of enhanced green fluorescence protein (EGFP)in ribosomes in combination with RNA purification.

翻译核糖体亲性纯化(TRAP),用于从体内内皮细胞中分离RNA

Many studies have been limited to using in vitro cellular assays and whole tissues or isolating of specific cell types from animals for in vitro analysis ...

RNA Blot Analysis for the Detection and Quantification of Plant MicroRNAs

MicroRNAs (miRNAs) are a class of endogenously expressed non-coding, ~21 nt small RNAs involved in the regulation of gene expression in both plants and animals. Most miRNAs act as negative switches of gene expression targeting key genes. In plants, primary miRNAs (pri-miRNAs) transcripts are generated by RNA polymerase II, and they form varying lengths of stable stem-loop structures called pre-miRNAs. An endonuclease, Dicer-like1, processes the pre-miRNAs into miRNA-miRNA* duplexes. One of the strands from miRNA-miRNA* duplex is selected and loaded onto Argonaute 1 protein or its homologs to mediate the cleavage of target mRNAs. Although miRNAs are key signaling molecules, their detection is often carried out by less than optimal PCR-based methods instead of a sensitive northern blot analysis. We describe a simple, reliable, and extremely sensitive northern method that is ideal for the quantification of miRNA levels with very high sensitivity, literally from any plant tissue. Additionally, this method can be used to confirm the size, stability and the abundance of miRNAs and their precursors.

This method demonstrates use of the northern hybridization technique to detect miRNAs from total RNA extract.

用于植物微RNA检测和定量的RNA斑点分析

MicroRNAs (miRNAs) are a class of endogenously expressed non-coding, ~21 nt small RNAs involved in the regulation of gene expression in both plants and ...

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.