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
Summary
在这里，我们描述了一个分步策略，用于隔离小型RNA、丰富微RNA和为高通量测序准备样本。然后，我们介绍如何使用开源工具处理序列读取并将其与微RNA对齐。
Abstract
一半的人类记录被认为是由微RNA调节的。因此，量化微RNA表达可以揭示疾病状态的基本机制，并提供治疗靶点和生物标志物。在这里，我们将详细介绍如何准确量化微RNA。简而言之，该方法描述了分离微RNA、将它们与适合高通量测序的适配器、放大最终产品以及准备样本库。然后，我们解释如何将获取的测序读数与微RNA发夹对齐，并量化、规范化和计算其差分表达。多功能且坚固，这种结合的实验工作流程和生物信息分析使用户能够从组织提取开始，完成微RNA定量。
Introduction
人类基因组2中首次发现于1993年1，现在估计有近2000个微RNA。微RNA是小型非编码RNA，通常为21-24个核苷酸长。它们是基因表达的转录后调节器，通常结合目标基因的3个未翻译区域（3-UTR）的互补位点，以抑制蛋白质表达和降解mRNA。量化微RNA可以给基因表达提供有价值的见解，并为此开发了几种协议3。
我们开发了一种定义、可重复和长期的协议，用于小型RNA测序，以及使用开源生物信息学工具分析规范化读取。重要的是，我们的协议能够同时识别内源性微RNA和外源交付的构造，从而产生类似微RNA的物种，同时最大限度地减少到其他小RNA物种（包括核糖体RNA）的读数。rRNA），转移RNA衍生的小RNA（tsRNA），重复衍生的小RNA和mRNA降解产物。幸运的是，微RNA是5-磷酸化和2-3羟基化4，一个功能，可以利用它们从这些其他小RNA和mRNA降解产物分离。对于微RNA克隆和测序，存在若干商业选择，这些选择往往更快、更容易进行多路复用;然而，试剂盒的专有特性及其频繁的修改使得比较样品运行具有挑战性。我们的策略通过丙烯酰胺和阿甘蔗凝胶纯化步骤，仅优化收集正确大小的微RNA。在此协议中，我们还描述了使用开源工具将序列读取与微RNA对齐的过程。这套说明对于信息学新手用户特别有用，无论我们使用的是库准备方法还是商业方法。
该协议已用于几个已发表的研究。例如，它被用来识别Dicer酶在距离茎环结构内部回路两个核苷酸的距离上切割小发夹RNA的机制，即所谓的”循环计数规则”5。我们还遵循这些方法，以识别从重组的阿德诺相关病毒载体 （rAAV） 表达的小发夹 RNA （shRNA） 的相对丰度，以识别在肝脏之前可以耐受的 shRNA 表达阈值毒性与过量shRNA表达6相关。利用这个协议，我们还确定了肝脏中的微RNA，这些微RNA对缺乏微RNA-122（一种高度表达的肝微RNA）作出反应，同时也确定了这种微RNA7的降解模式。由于我们在众多实验中一致地使用了我们的协议，因此我们能够纵向观察样品制剂，并且发现没有明显的批次效应。
在共享该协议时，我们的目标是使用户能够使用负担得起的设备和试剂以及免费的生物信息学工具，在几乎任何组织或细胞系中生成高质量、可重复的微RNA定量。
Protocol
动物实验得到了华盛顿大学机构动物护理和使用委员会的授权。 小型RNA库制备 1. RNA分离 使用标准RNA分离试剂或用于微RNA的试剂盒从生物源分离RNA。对于组织，最好从样品开始，用液氮冷冻样品，然后用预冷冻砂浆和虫子将样品冷冻到粉末中。 在可量化RNA并提供RNA完整性数（RIN）的仪器上测量每个样品的RNA完整性。…
Representative Results
图书馆准备中涉及的步骤图图2概述了小RNA提取、测序和对齐的总体原理图。采集了一只雄性小鼠和一只雌性小鼠的肝脏样本，并冷冻在液氮中。提取总RNA并评估质量和浓度。 小RNA测序产生足够的RNA测序从两个独立的RNA提取中提取的3μgRNA被用作小RNA测序的起始材料。样?…
Discussion
尽管20多年前鉴定了微RNA，但微RNA测序过程仍然十分费力，需要专门的设备，阻碍了实验室常规采用内部协议14。其他技术可以同时评估微RNA，如微RNA微阵列和多路复用表达面板;然而，这些方法是有限的，因为它们只量化其探针集中存在的微RNA。因此，他们忽略了小RNA测序的重要特征，如新微RNA的鉴定，以及微RNA等形式-核苷变化，这些改变可以产生重要的生物?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
我们要感谢安德鲁·火和马克·凯实验室的成员的指导和建议。
Materials
| 100 bp DNA ladder | NEB | N3231 | |
| 19:1 bis-acrylamide | Millipore Sigma | A9926 | |
| 25 bp DNA step ladder | Promega | G4511 | |
| Acid phenol/chloroform | ThermoFisher | AM9720 | |
| Acrylamide RNA loading dye | ThermoFisher | R0641 | |
| Ammonium persulfate (APS) | Biorad | 161-0700 | |
| Bioanalyzer instrument | Agilent | G2991AA | For assessing RNA quality and concentration |
| Chloroform | Fisher Scientific | C298-500 | |
| Ethanol (100%) | Sigma | E7023 | |
| Gel Loading Buffer II | ThermoFisher | AM8547 | |
| GlycoBlue | ThermoFisher | AM9516 | Blue color helps in visualizing pellet |
| HCl | Sigma | 320331 | |
| KOH | Sigma | P5958 | |
| Maxi Vertical Gel Box 20 x 20cm | Genesee | 45-109 | |
| miRVana microRNA isolation kit | ThermoFisher | AM1560 | |
| miSeq system | Illumina | SY-410-1003 | For generating small RNA sequencing data |
| NaCl | Fisher Scientific | S271-500 | |
| Nusieve low-melting agarose | Lonza | 50081 | |
| Parafilm (laboratory sealing film) | Millipore Sigma | P7793 | |
| Poly-ethylene glycol 8000 | NEB | included with M0204 | |
| ProtoScript II First strand cDNA Synthesis Kit | NEB | E6560S | |
| QIAquick Gel Extraction kit | Qiagen | 28704 | |
| Qubit Fluorometer | ThermoFisher | Q33226 | For quantifying DNA concentration |
| Qubit RNA HS Assay kit | ThermoFisher | Q32855 | |
| Razor Blades | Fisher Scientific | 12640 | |
| Siliconized Low-Retention 1.5 ml tubes | Fisher Scientific | 02-681-331 | |
| T4 RNA ligase 1 | NEB | M0204 | |
| T4 RNA Ligase 2, truncated | NEB | M0242S | |
| TapeStation | Agilent | G2939BA | For assessing RNA quality and concentration |
| Taq DNA Polymerase | NEB | M0273X | |
| TEMED | Biorad | 161-0800 | |
| Tris Base pH 7.5 | Sigma | 10708976001 | |
| Tris-buffered EDTA | Sigma | T9285 | |
| Trizol | ThermoFisher | 15596026 | |
| UltraPure Ethidium bromide (10 mg/ml) | Invitrogen | 15585-011 | |
| Universal miRNA cloning linker | NEB | S1315S | |
| Urea | Sigma | U5378 |
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Cite This Article
Course, M. M., Gudsnuk, K., Valdmanis, P. N. A Complete Pipeline for Isolating and Sequencing MicroRNAs, and Analyzing Them Using Open Source Tools. J. Vis. Exp. (150), e59901, doi:10.3791/59901 (2019).