高密度脂蛋白的分离的非编码小RNA定量
Summary
This protocol describes the isolation and quantification of high-density lipoprotein small RNAs.
Abstract
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。
Introduction
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。
Protocol
Representative Results
Discussion
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…
Declarações
The authors have nothing to disclose.
Acknowledgements
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.
Materials
| Ultracentrifuge | Beckman Coulter | A99839 | Optima XPN-80 |
| Ultracentrifuge Rotor | Beckman Coulter | 331362 | SW-41Ti |
| AKTA Pure FPLC System | GE Healthcare | 29018224 | |
| 3X FPLC Superdex 200 Increase Columns In-line | GE Healthcare | 28990944 | 10/300 gl |
| SynergyMx | BioTek Instruments | 7191000 | |
| Tabletop centrifuge | Thermo Scientific | 75004525 | Sorvall ST40R |
| Refrigerated centrifuge | Eppendorf | 22629867 | 5417R (purchased through USA Scientific) |
| Microfuge | USA Scientific | 2631-0006 | |
| PippenPrep | Sage Science | PIP0001 | |
| 2100 Bioanalyzer | Agilent | G2938B | |
| High Sensitivity DNA Assay | Agilent | 5067-4626 | |
| Sequencing Library qPCR Quantification Kit | Illumina | SY-930-1010 | |
| ProFlex Thermal Cycler | Applied Biosystems | 4484073 | |
| QuantStudio 12k Flex | Applied Biosystems | 4471134 | |
| EpMotion Robot | Eppendorf | 960000111 | 5070 |
| Ultra-clear centrifuge tubes | Beckman Coulter | 344059 | |
| Potassium Bromide | Fisher Chemicals | P205-500 | |
| 15 mL conical tube | Thermo Scientific | 339650 | |
| Micro-centrifugal filters 0.45µm | Millipore | UFC30HV00 | |
| Micro-centrifugal filters 0.22µm | Millipore | UFC30GV00 | |
| miRNAEasy Total RNA Isolation Kits | Qiagen | 217004 | |
| Total Cholesterol colormetric kit | Cliniqa (Raichem) | R80035 | |
| 10,000 m.w. cut-off centrifugation filter | Amicon | UFC801024 | purchased through Millipore |
| PCR strip tubes | Axygen | PCR-0208-C | purchased through Fisher |
| microRNA RT kit | Life Technologies | 4366597 | For 1000 reactions |
| PCR master mix | Life Technologies | 4440041 | 50 mL bottle |
| Pierce BCA kit | Thermo Scientific | 23225 | |
| Clean and Concentrator Kit | Zymo | D4014 | |
| Dialysis tubing | Spectrum Labs | 132118 | purchased through Fisher |
| bcl2fastq2 | Illumina | n/a | Software |
| Cutadapt | https://github.com/marcelm/cutadapt | n/a | Software |
| NGSPERL | github.com/shengqh/ngsperl | n/a | Software |
| CQSTools | github.com/shengqh/CQS.Tools | n/a | Software |
| Bowtie 1.1.2 | http://bowtie-bio.sourceforge.net | n/a | Software |
| GeneSpringGX13.1.1 | Agilent | n/a | Software |
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