小RNA分子的高效结扎的微小RNA定量高通量测序
Summary
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.
Abstract
miRNA的克隆和高通量测序，所谓的miR-SEQ，独树一帜的全基因组范围的方法来量化的miRNA与单核苷酸分辨率。这种技术捕获的miRNAs通过连接3'和5'寡核苷酸衔接头到miRNA分子，并允许从头 miRNA的发现。具有强大的新一代测序平台的耦合中，miR-SEQ一直在miRNA的生物学研究。然而，寡核苷酸连接的步骤介绍显著的偏见阻碍的miR-SEQ被用作一个精确的定量工具。以往的研究表明，在当前的miR-SEQ方法的偏见往往会导致不准确的miRNA定量与错误的高达1000倍的某些miRNA的1,2。要解决这些偏见，通过RNA结扎传授，我们已经开发出一种小RNA结扎方法导致95％以上的为3'和5'连接步骤连接效率。这个基准测试IM证明文库构建方法，使用等摩尔或差分混合的合成的miRNA，始终如一地产生具有与预期值小于2倍的偏差读出的数字。此外，这种高效率的miR-SEQ方法允许从体内总RNA样品2精确的全基因组miRNA表达谱。
Introduction
高通量测序基础的方法已被广泛应用到许多生物样品近年来大大扩大我们的生物系统3,4的分子复杂性的理解。然而，制备RNA样品进行高通量测序的常赋予固有的就业方法的具体偏差，限制了这些强大的技术的潜在效用。这些具体的方法偏差得到了很好的证明了结扎为主，小RNA库准备1,2,5,6。这些偏见导致1000倍的变化读取号码等摩尔合成的miRNA，使得miRNA的丰推断，从测序数据疯狂可变且容易出错。
研究着重于噬菌体衍生的T4 RNA连接酶的性质已经证明，酶表现出的基于核苷酸的偏好7，它体现在高通量测序实验作为偏置库<sUP> 1,2,8。为了最小化被RNA连接酶所赋予的偏见，多种策略已经使用;大分子拥挤9中 ，随机化是近端结扎部位6的适配器上的碱基序列，以及使用高浓度的结扎适配器2。通过这三种方法的组合，我们开发了一个工作流的小RNA文库的高通量测序（ 图1）兼容的无偏制剂。对于目前的协议和我们的优化方法进行直接的比较，请参阅最近的一份报告2。这种优化的方法产生的大于95％的在两个3'和5'步骤结扎的效率，并允许小分子RNA合成和生物样品2的无偏结扎。
Protocol
Representative Results
Discussion
本文所描述的方法利用了几个关键变量最大化连接效率，即高浓度的PEG，用随机接头的，与高浓度的接头2,6,9的。这种方法可以从总RNA样品2可靠的定量测序文库。我们所进行的输入的RNA多步滴定，并得出结论认为，前面的方法是最适合的总RNA的量在1-8微克的范围（数据未显示）。当在10-500纳克范围的量使用，大部分的读取空间由适配器多联体，并从其中所述RNA连接酶被纯化细菌序…
Disclosures
The authors have nothing to disclose.
Acknowledgements
The authors would like to thank members of the Yi laboratory especially Zhaojie Zhang for fruitful discussions regarding linker design and ligation efficiencies, as well as the American Cancer Society for supporting this work through a postdoctoral fellowship (#125209) to J.E.L. Research reported in this publication was also supported by the National Institute of Arthritis and Musculoskeletal and Skin Diseases of the National Institutes of Health under Award Number R01AR059697 (to R.Y.) and a research grant from the Linda Crnic Institute for Down Syndrome. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
Materials
| Name of Reagent/ Equipment | Company | Catalog Number | Comments/Description |
| 3' Linker (5' phosphorylated, 3' blocked) | Integrated DNA Technologies | custom | |
| 5' Linker | Integrated DNA Technologies | custom | 5' blocked, HPLC Purified |
| T4RNL2 (1-249 K227Q) | New England Biolabs | M0351S | Specialized for ligation of pre-adenylated DNA adapters |
| 10X Ligation Buffer (without ATP) | New England Biolabs | Included with M0351S | |
| 10X Ligation Buffer (with ATP) | New England Biolabs | Included with M0204L | |
| RNaseOUT | Invitrogen | 10777-019 | |
| Polyethylene Glycol (mol. Wt. 8000) | New England Biolabs | Included with M0204L | |
| Nuclease-free water | Ambion | AM9937 | We have found water collected from a distillation apparatus to be of equvalent quality. |
| T4RNL1 | New England Biolabs | M0204L | |
| Superscript III RT kit | Invitrogen | 18080-051 | |
| Phusion PCR kit | New England Biolabs | M0530S | |
| Illumina RP1 Primer | Integrated DNA Technologies | custom | Sequence information available from Illumina |
| Illumina RT Primer | Integrated DNA Technologies | custom | Sequence information available from Illumina |
| Illumina Index Primer(s) | Integrated DNA Technologies | custom | Sequence information available from Illumina |
| 40% Acrylamide | Fisher Scientific | BP14081 | |
| Urea | Sigma Aldrich | U6504 | |
| Ammonium persulfate | Sigma Aldrich | A3678 | |
| Tetramethyethylenediamine (TEMED) | Sigma Aldrich | T9281 | |
| 2X Denaturing RNA loading buffer | New England Biolabs | Included with M0351S | |
| Razor blades | VWR | 55411-050 | |
| SpinX Centricon Tubes | Costar | CLS8161 | |
| Low Retention Microfuge tubes | Fisher Scientific | 02-681-320 | |
| Sybr Gold | Invitrogen | S-11494 | |
| Adenylation Kit | New England Biolabs | E2610L |
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