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Summary
Abstract
Introduction
Protocol
Results
Discussion
Disclosures
Acknowledgements
Materials
References
자동 번역
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생물학

增强Northern印迹检测小RNA种类的<em>果蝇</em

Published: August 21, 2014
doi:
10.3791/51814
,
1Département de Génomique Fonctionnelle et Cancer,Institut de Génétique et de Biologie Moléculaire et Cellulaire, 2Center for Life NanoScience,Istituto Italiano di Tecnologia

Summary

本刊物的目的是可视化和探讨手术步骤，从果蝇的RNA提取的增强Northern杂交协议的果蝇胚胎，细胞和组织。这个协议是用于有效地检测小RNA种类是特别有用的。

Abstract

在过去的几十年目睹身边的基因表达调控机制的科学兴趣在RNA水平的爆炸。分子生物学的这个分支已经大大助长了非编码RNA作为主要参与者的转录后调控的发现。这样一种革命性透视一直伴随着与由强大的技术开发的仿形短的RNA表达触发，无论是在高通量水平（全基因组鉴定）或单候补分析（特定物种的稳态积累）。虽然国家的最先进的几种策略是目前可用于定量或可视化等逃离分子，RNA印迹分析仍然是分子生物学的RNA表达的即时和准确的评估资格的办法。它代表了对更复杂的，昂贵的技术，在许多情况下，应用程序的第一步，仍然是一个优先的方法来轻松地深入了解成RNA生物学。在这里，我们概述一个有效的协议（增强Northern杂交），用于检测果蝇弱表达的microRNA（或其他小调控RNA种类）全果蝇胚胎，人工解剖幼虫/成人组织或体外培养的细胞。是必需的RNA的一个非常有限的量，并利用材料从流式细胞仪，分离的细胞可以是也可以设想。

Introduction

RNA生物化学经历了惊人的进步，在过去的1年。我们的基因表达的对照RNA的潜在的理解已经爆裂通过强大的非编码核糖调节2的识别，通过新颖的基于RNA的调节机制3,4的发现和利用已公知的转录后事件更深的表征5。总之这些研究使RNA的生物学大大使场景，成为了目前的科学景观的一个主要研究课题。特别是近年来，我们越来越的“RNA世界”的分子神经生物学6，在现代生命科学中最具活力的研究领域之一的普遍冲击感。在过去的一个世纪的最后十年，整体科学的情景已经被彻底改变了小调控RNA 9,10的RNA干扰7,8的发现和特别是关于微小RNA 11，内源性表达牵连的几乎所有细胞功能基因表达的多效性和组合调节控制的小非编码RNA。

近10年后的miRNA最初发现在秀丽隐杆线虫由安布罗斯和Ruvkun的实验室，重新关注被拒绝到外地时，在果蝇和人类细胞以及12-15确定了高数量的miRNA。从那时起，这要归功于灵活的转基因方法， 果蝇已经站在了一个宝贵的生物背景的深入研究miRNA的生物合成和活性。 果蝇的miRNA揭示了不同的功能的昆虫特异性或进化上保守的过程，老化代谢，信号转导通路跨越，行为，当然，神经发生。沿着这个方向，我们最近推出了有趣的合作一个新的链接16rrelation主基因GCM /滑翔和RNA的通路之间发生。飞转录因子GCM /滑翔17-19构成的细胞命运的决定因素，这决定了胶质细胞与神经元命运的选择多能飞的神经前体20一个独特的例子。二十年对这个话题很长的研究已经清楚地强调了基因表达调控多和重叠的投入发生了融合GCM /滑行 21-28建立所需的神经发育过程中神经的平衡和神经胶质同行之间的微妙比例的阈值水平。

我们发现，通过DMEL-miR279目标，监管是进一步控制水平有助于转录后微调的GCM /滑动式16。从全球来看，这些研究都行需要具体的方法改进：沿几年，一些技术最初开发分析TRADitional的RNA被转化为定量小的非编码RNA，等作为RNA酶保护测定，cDNA芯片29-31，实时PCR的方法32-35和测序36,37。在另一边，技术方法校准助长了持续进展的领域。

Northern印迹法（NB，或RNA凝胶印迹法）构成了一个有代表性的实例：它主要用于型材RNA的积累，因为它确保了表达水平的定量和尺寸确定。然而，该方法的特性差的灵敏度被限制时，它是被应用到低丰度的基因表达细调谐器，像短的RNA。甲不利后果是大量的总RNA，这使得难以应用到特定的生物样品的要求。对于这样的理由，小分子RNA检测特定NB变种已经发展38-40：我们采取了一种改进的NB PROC的优势edure 41（ENB，增强Northern印迹），同时阐明DMEL-miR279和GCM /滑翔之间的上述相互作用。

此方法依赖于基于碳二亚胺衍生物的活性的化学交联步骤[1 – 乙基 – 3（3-二甲基氨基丙基）碳化二亚胺，EDC]固定核酸到固体支持物。碳二亚胺是已知催化的酰胺键的活化羧基或磷酸基团和胺基团42之间形成一个多功能的交联剂。此属性可通过其单磷酸5'-羟基基团被利用共价耦合的小分子RNA，以氨基基团在尼龙膜的表面上。所得的附接结构增加了固定化的核酸的可访问性，反过来，探针-靶杂交，这导致显着的检测增强43的效率。

该技术假设特定relevancE在果蝇的分子生物学研究，通过这些新颖独特的类小非编码RNA的产生是新兴的44。在这些中，rasiRNAs 45,46构成PIWI相互作用RNA（了piRNA 47），参与序列特异性基因沉默的特异性亚型。此方法的执行细节中充分描述和可视化在下文中，相对于微小RNA DMEL-miR279和DMEL-miR286的分析，并在第一时间，有rasiRNA，rasi4的。我们推走极端此方法，该方法允许我们从RNA（小于1微克）的最小量的揭示表达很差目标。

Protocol

1，样品采集分离半粘合施耐德2细胞（1-5×10 6个细胞的平均值），通过移液并通过离心（100×g离心1分钟）收获它们。如果在体外培养的贴壁细胞，trypsinize他们在标准条件下，或者直接从板上将它们收集到的RNA提取试剂的便利量，用细胞刮刀的帮助。 对于胚胎收集，成长果蝇株苍蝇笼子里，让胚胎积聚在产蛋板。在蒸馏水画笔（DH 2 O） 的胚胎?…

Representative Results

通过下面的文本中， 如图1中所描述和示意性地表示的整体过程中，我们评估不同来源的复杂的RNA样品的小RNA的表达。在图1中给出的实验中，RNA从果蝇施耐德2细胞通过蛋白酶K提取，检查其完整性分离（可选步骤：变性琼脂糖凝胶分离），并在双加载。凝胶的一半印迹在中性膜和化学交联的EDC;第二半被转移上带正电的尼龙过滤器，然后UV交联的（1.2×10 5个μ…

Discussion

虽然我们的阅历相互交织，通过它的RNA调节神经的升值​​不断增加，基于RNA的设计电路影响神经干细胞生物学，神经细胞的分化/功能集成，神经病变和癌症的发展机理的影响尚未被探索。这样的网络通过王国的维护使得遗传系统的潜力作为果蝇有助于揭开未开发的细胞途径，其中短的非编码RNA和转录因子被认为是主要的分子球员。在此视图中，候选小分子RNA表达水平的概要是propaedeutic到…

Disclosures

The authors have nothing to disclose.

Acknowledgements

这项工作是由国家研究所德拉桑特等德拉RECHERCHEMédicale的支持下，该中心的法国国家科学研究中，圣圣斯特拉斯堡总医院斯特拉斯堡，协会倒拉RECHERCHE河畔勒癌，国立研究所杜癌症，法新社的国立德拉RECHERCHE和地区阿尔萨斯。

彼得拉内韦一直支持基金会倒拉RECHERCHEMédicale。目前，他是一个因诺琴蒂基金会意大利语TECNOLOGIA（IIT）的奖学金的获得者。出版费用由Neurex网络支持（TriNeuron – 计划区域间四上莱茵省）

Materials

FINAL COMPOSITION/NAME COMPANY CATALOGUE (Stock) COMMENTS
GENERAL REQUIREMENT
Centrifuge, 5418 Eppendorf 5418 000.017
Decontaminant, RNase Away Sigma-Aldrich 83931 Apply on glass/plasticware, wipe and rinse
RNase inhibitor, DEPC Sigma-Aldrich D5758 Hazardous //// 1609-47-8
0.1 % DEPC suspension Incubate 2 hr at 37 ˚C, then sterilize by autoclave
SAMPLE PREPARATION
Stereo Microscope, M60 Leica 
1X PBS Buffer
137 mM NaCl Sigma-Aldrich S3014 7647-14-5  
2.7 mM KCl Sigma-Aldrich P9541 7447-40-7 
10 mM Na2HPO4 Sigma-Aldrich S3264 7558-79-4 
1.8 mM KH2PO4 Sigma-Aldrich P9791 7778-77-0 
RNA EXTRACTION/ANALYSIS
UV-Vis Spectrophotometer Thermo Scientific Nanodrop 2000
Gel Imager, ChemiDoc XRS+ Biorad 170-8265
Horizontal Electrophoresys, Mini-Sub Cell GT BioRad 170-4487EDU
RNA extraction reagent, TRIzol Reagent Life Technologies 15596026 Hazardous
PCA (25:24:1), 1:1 for extraction Sigma-Aldrich 77617 136112-00-0
Chlorophorm, 1:1 for extraction Sigma-Aldrich C2432 67-66-3  
EtOH, 2.5 vol. for precipitation, 70% for washing Sigma-Aldrich 59844 64-17-5 
Gene Ruler 1Kb DNA Ladder Thermo Scientific SM0311
Stop Mix
300 mM NaOAc, pH 5.5 Sigma-Aldrich S2889 127-09-3
1x RSB Solution
2% sodium dodecyl sulfate (SDS) Sigma-Aldrich 71736 Never cool down SDS to avoid  precipitation //// 151-21-3
2 mg/ml Proteinase K Roche Applied Science 0-3115828001 EC 3.4.21.64 9
10X Reticulocyte Standard Buffer (RSB)
100 mM Tris-Cl, pH 7.4 Sigma-Aldrich T5941 1185-53-1  
100 mM NaCl Sigma-Aldrich S3014 7647-14-5  
30 mM MgCl2 Sigma-Aldrich M8266 7786-30-3
Denaturing Agarose Gel
1.2% Agarose Sigma-Aldrich A9539 Let the gel solidify for at least 1 hr //// 9012-36-2
1x MOPS Buffer Add when T < 60 ˚C
3% formaldehayde Sigma-Aldrich F8775 Hazardous. Add when T < 60 ˚C //// 50-00-0
10X MOPS Buffer
0.2 M MOPS sodium salt, pH 7 Sigma-Aldrich M9381 Do not confuse MOPS sodium salt with MOPS //// 71119-22-7
Agarose Loading Dye
1x MOPS Buffer
3% formaldehyde Sigma-Aldrich F8775 Hazardous //// 50-00-0
50% formamide Sigma-Aldrich 47670 Hazardous //// 75-12-7
Ethidium bromide (EtBr) 30 μg/ml Sigma-Aldrich E1510 Hazardous //// 1239-45-8
Bromophenol blue  Sigma-Aldrich B0126 115-39-9 
SMALL RNA FRACTIONATION
Flat gel loading tips Life Technologies LC1002
Savant SpeedVac Concentrator Thermo Scientific  DNA120
Vertical Electrophoresis, Mini-PROTEAN Tetra Cell  Biorad 165-8000
Denaturing Acrilamide Gel
10% acrylamide/bis-acrylamide (19:1) Sigma-Aldrich A3449 Hazardous. Let the gel polymerizefor 45 min before use
1x TBE Buffer (or 1x MOPS Buffer)
7 M urea Sigma-Aldrich U6504 57-13-6
0.1% ammonium persulfate Sigma-Aldrich 215589 7727-54-0 
0.1% TEMED Sigma-Aldrich T9281 110-18-9
Gene Ruler Ultra Low Range DNA Ladder (10 bp-step) Thermo Scientific SM1211 Label 0.1 μg, as described in the "probe labeling" reaction
Acrylamide Blue Dye 
50% formamide Sigma-Aldrich 47670 Hazardous //// 75-12-7
Bromophenol blue Sigma-Aldrich B0126 115-39-9 
Running Buffer (RB)
1x MOPS Buffer 
Alternative RB: 1x TBE Buffer
1x TBE Buffer
5X TBE
445 mM Tris-base Sigma-Aldrich T1503 77-86-1 
445 mM boric acid Sigma-Aldrich B7901 10043-35-3
20 mM EDTA Sigma-Aldrich EDS 60-00-4  
Gel Staining Solution
Ethidium bromide 0.5 μg/ml Sigma-Aldrich E1510 Hazardous //// 1239-45-8
1x RB Stain for 15 min with EtBr, destain for 15 min w/o EtBr
BLOTTING
3MM Whatman paper Sigma-Aldrich Z270849
Neutral Nylon Membrane, Hybond NX GE Healthcare Life Science RPN203T Photosensitive
CROSSLINKING
Crosslinking Solution (XLS)
1% 1-methylimidazole (v/v) Sigma-Aldrich 336092 Always prepare a fresh aliquot of XLS //// 616-47-7
12.5 mM HCl Sigma-Aldrich H1758 Hazardous //// 7647-01-0
3.1% EDC (w/v) Sigma-Aldrich 3450 25952-53-8  
(PRE)-HYBRIDIZATION
Liquid Scintillation Counter  Beckmann LS 6500
Sheared Salmon Sperm, 0.1 mg/ml  Life Technologies AM9680
rasi4 antisense probe 5' CGGUGUUCGACAGUUCCUCGGG -3'
5s-rRNA antisense probe 5'-CAACACGCGGTGTTCCCAAGCCG-3'
Dmel-miR279 antisense probe 5'-TTAATGAGTGTGGATCTAGTCA-3'
Dmel-miR286 antisense probe 5'-AGCACGAGTGTTCGGTCTAGTCA-3'
Purification Kit, Illustra MicroSpin G-25 Columns GE Healthcare Life Science 27-5325-01
(Pre)-Hybridization Solution (HS)
6x SSPE Pre-warm HS at 37 ˚C before use
0.5% SDS Sigma-Aldrich 71736 151-21-3 
5x Denhardt's Solution
20X SSPE
3.6 M NaCl Sigma-Aldrich S3014 7647-14-5  
0.2 M sodium phosphate Sigma-Aldrich 342483 7601-54-9  
20 mM EDTA (pH 8.0) Sigma-Aldrich EDS  60-00-4  
50x Denhardt's Solution
1% Ficoll 400 (w/v) Sigma-Aldrich F2637 26873-85-8
1% Polyvinylpyrrolidone Sigma-Aldrich PVP40 9003-39-8
1% BSA Sigma-Aldrich A7906 9048-46-8  
1X TEN Buffer
10 mM Tris-Cl (pH 8.0) Sigma-Aldrich T1503 77-86-1 
100 mM NaCl Sigma-Aldrich S3014 7647-14-5  
1 mM EDTA (pH 8.0) Sigma-Aldrich EDS  60-00-4  
Probe Labelling
0.4 μM oligonucleotide
1.5 μl Ci/μl [γ-32P] ATP Perkin Elmer NEG002A Hazardous //// 51963-61-2
1x T4 Polynucleotide Kinase Buffer Biolabs B0201
T4 Polynucleotide Kinase 0.5 units/μl Biolabs M0201 EC 2.7.1.78
30 min at 37 ˚C, stop by 20 mM EDTA
WASHING
Geiger Mueller Detectors Canberra Industries MCB2/CPS – EM77021
Washing Buffer (WB), 2x SSPE
Stringent Washing Buffers
0.2x SSPE
2x SSPE, 0.5% SDS
0.2x SSPE, 0.5% SDS
SIGNAL DETECTION
PharosFX Plus System Biorad 170-9460
Image J Freely available
Quantity One Biorad
X-ray films , BioMax XAR Sigma-Aldrich F5888 Photosensitive
Developer for X-ray films Sigma-Aldrich P7042
Fixer for X-ray films Sigma-Aldrich P7167
STRIPPING
Stripping Solution
10 mM Tris-HCl pH 8.8 Sigma-Aldrich T1503 77-86-1 
5 mM EDTA Sigma-Aldrich EDS  60-00-4  
0.1% SDS Sigma-Aldrich 71736 Add SDS after boiling to avoid foaming //// 151-21-3
DOWNLOAD MATERIALS LIST

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Enhanced Northern BlotSmall RNAMicroRNADrosophila MelanogasterGene ExpressionPost-transcriptional RegulationNoncoding RNARNA ProfilingRNA DetectionMolecular BiologyEmbryoCell Culture

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Laneve, P., Giangrande, A. Enhanced Northern Blot Detection of Small RNA Species in Drosophila Melanogaster. J. Vis. Exp. (90), e51814, doi:10.3791/51814 (2014).
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