人骨髓间充质干细胞细胞外微 rna 的分离、测序和分析
Summary
该协议演示了如何从细胞培养培养基中纯化细胞外微 rna, 用于小型 rna 文库构建和下一代测序。描述了各种质量控制检查点, 以使读者了解在处理低输入样本 (如 exrna) 时的预期结果。
Abstract
细胞外和循环 rna (exrna) 是由身体的许多细胞类型产生的, 并存在于许多体液中, 如唾液、血浆、血清、牛奶和尿液。这些 rna 的一个子集是转录后的调节剂–微 rna (mirna)。为了描述特定细胞类型产生的 mirna, 体外培养系统可用于从一个细胞子集中提取和分析 exrna。骨髓间充质干细胞的分泌因子与减轻多种疾病有关, 并作为本研究的体外模型系统。本文介绍了小 rna 的采集、纯化和细胞外 mirna 序列库的生成过程。培养基中的 exrna 与细胞 rna 的不同之处在于 rna 样本较低, 这就需要优化程序。该协议为来自培养基的小 exrna 测序提供了全面的指南, 显示了 exrna 纯化和测序过程中每个步骤的质量控制检查点。
Introduction
细胞外和循环 rna (exrna) 存在于各种体液中, 并对 rnase1, 2 具有耐药性。它们的高丰度、稳定性和易用性作为诊断和预后标志物 3, 对临床评估很有吸引力。外生殖器的运输方式包括细胞外囊泡 (ev), 与脂蛋白 (如高密度脂蛋白) 的联系;高密度脂蛋白) 和核糖核酸蛋白复合物 (如与 Argonaute2 复合物)4。
exrna 的一个子集是微 rna (mirna), 它们是大约22码的小非编码 rna, 调节转录后的基因表达。前 mirna 与细胞通讯和细胞稳态调节有关。例如, hdl 将前 mir-223 传递给内皮细胞, 以抑制细胞间粘附分子 1 (icam-1) 和炎症6。有趣的是, mir-223 也是由细胞外泡从白细胞转移到肺癌细胞, 编程它们采取更具侵入性的表型 7.因此, 从各种体液和细胞培养培养基的前 mirna 转录组将大大提高我们对前 mirna 信号的理解。
小 rna 测序 (小 rna seq) 是一个强大的工具, 可用于了解小 rna 的转录组学。不仅可以在不同表达的已知 rna 中比较不同的样品, 而且还可以检测和表征新的小 rna。因此, 在不同条件下识别差异表达的 mirna 也是一种鲁棒性。然而, 小 rna seq 的障碍之一是难以从低外显侧 nna 输入液 (如脑脊液、唾液、尿液、牛奶、血清和培养基) 产生小的 rna seq 库。来自 illumina 的 truseq 小型 rna 库准备协议需要大约1μg 的高质量总 rna, 而 nebnext 小型 rna 库准备集协议来自新英格兰 biolabs需要100 ng-1μg 的 rna 8,9。对于传统的 uv-vis 分光光度计, 这些样品的总 rna 通常低于检测极限。
来自体液的前 mirna 具有潜在的良好预后和诊断标志物。然而, 为了研究功能效应或确定特定的前 mirna 的起源, 经常使用细胞培养系统。间充质干细胞 (mscs) 已经被广泛的研究, 因为他们的电动车已涉及减轻许多疾病, 包括心肌梗死, 阿尔茨海默病和移植物抗宿主病10。在这里, 我们演示了从骨髓衍生的 mscs (bmscs) 中纯化前 mirna 的方法, 以及用于优化小型 rna 库构建、测序和数据分析的具体步骤 (图 1)。
Protocol
Representative Results
Discussion
在这里, 我们描述了一种用于下一代 exrna 测序的协议, 该协议支持从低输入样本进行差异表达式分析。坚持电动汽车和 exRNA 分离的特定协议是很重要的, 因为即使是微小的改变 (即超离心步骤或转子类型的变化) 也会影响转录组和 mirna 水平 13,14.因此, 无论 exrna 是如何分离的, 重要的是应用相同的实验和生物信息程序的所有样本在实验中能够比较的结果。<…
Disclosures
The authors have nothing to disclose.
Acknowledgements
我们感谢克劳斯·布斯先生和伊纳诺的丽塔·罗森达尔女士的技术援助。特别感谢丹尼尔·奥特森博士允许我们频繁使用他的超离心机。这项研究得到了丹麦创新基金 (教育部项目) 的支持。
Materials
| Bone Marrow-Derived Mesenchymal Stem Cells | ATCC | PCS-500-012 | Cells used in this protocol was bought from ATCC |
| MSCGM BulletKit | Lonza | PT-3001 | Termed as Mesenchymal Stem Cell Growth Medium (MSC media) |
| Exosome-depleted FBS | Gibco | A2720801 | |
| ExRNA collecting media: MSCGM but with the FBS replaced by exosome-depleted FBS | |||
| Trypsin-EDTA | Gibco | 25200056 | |
| T175 Flask | Sarstedt | 833,912 | |
| Penicillin-Streptomycin | Gibco | 15140122 | |
| Phosphate Buffered Saline | Sigma | 806552 | |
| Ultracentrifuge | Beckman Coulter | ||
| Polycarbonate Bottle with Cap Assembly | Beckman Coulter | 355618 | |
| Beckman Coulter Type 60 Ti | Rotor used here | ||
| NucleoCounter | Chemometec | NC-3000 | Cell Counter |
| β-glycerophosphate | Calbiochem | 35675 | Components of the osteogenic differentiation media |
| Dexamethasone | Sigma | D4902-25MG | Components of the osteogenic differentiation media |
| 2-Phospho-L-ascorbic acid trisodium salt | Sigma | 49752-10G | Components of the osteogenic differentiation media |
| 1α,25-Dihydroxyvitamin D3 | Sigma | D1530-1MG | Components of the osteogenic differentiation media |
| miRNeasy Mini Kit | Qiagen | 217004 | miRNA and total RNA purification kit for step 4.8 |
| Agilent RNA 6000 Pico Kit | Agilent Technologies | 5067-1514 | Chip-based capillary electrophoresis machine and chips for RNA and DNA analysis |
| Agilent 2100 Bioanalyzer | Agilent Technologies | G2939BA | Chip-based capillary electrophoresis machine and chips for RNA and DNA analysis |
| Agilent High Sensitivity DNA Kit | Agilent Technologies | 5067-4626 | Chip-based capillary electrophoresis machine and chips for RNA and DNA analysis |
| KAPA Library Quantification Kits | Roche | KK4824 | Library quantification kit used here |
| TruSeq Small RNA Library Prep Kit -Set A (24 rxns) (Set A: indexes 1-12) | Illumina | RS-200-0012 | Small RNA library prepartion kit used in this protocol – used in step 5 |
| Pippin Prep | Sage Science | Automated DNA gel extractor used in this protocol; manual extraction can be done too | |
| MinElute PCR Purification Kit | Qiagen | 28004 | PCR purification in step 5.17 |
| FASTX_Toolkit | Cold Spring Harbor Lab | Trimming low-quality reads in step 6 | |
| cutadapt | Adaptor removal in step 6 | ||
| Bowtie | Mapping of clean reads in step 6 | ||
| Samtools | To make the expression profile in step 6 | ||
| Bedtools | To make the expression profile in step 6 |
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