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Neurowissenschaften

膜片钳后单细胞多重反转录聚合酶链反应

Published: June 20, 2018
doi:
10.3791/57627
, , ,
1UPMC Univ Paris 06, INSERM, CNRS, Neuroscience Paris Seine – Institut de Biologie Paris Seine (NPS – IBPS),Sorbonne Université

Summary

本协议描述了在膜片钳后执行单细胞多重反向转录聚合酶链反应所需的关键步骤和预防措施。该技术是一种简单有效的方法, 用于分析由膜片钳记录的单个细胞所确定的一组基因的表达谱。

Abstract

大脑皮层由多种细胞类型组成, 表现出多种形态、生理和分子特征。这种多样性阻碍了对这些细胞类型的识别和鉴定, 研究其特定功能的先决条件。本文介绍了多路单细胞反向转录聚合酶链反应 (rt-pcr) 协议, 它允许在切片的补丁钳记录后, 同时检测在单个细胞中的十个基因的表达。这种简单的方法可以进行形态学表征, 广泛适用于确定各种细胞类型的表型特征及其特定的细胞环境, 如血管附近。本协议的原则是用膜片钳技术记录一个细胞, 收获和反转转录其细胞质含量, 并通过多重 PCR 对预定义基因组的表达进行定性检测。它需要精心设计的 pcr 引物和细胞内贴钳解决方案兼容 rt-pcr。为了确保有选择和可靠的成绩单检测, 这项技术还需要适当的控制从细胞质采集到放大步骤。尽管这里讨论的预防措施必须严格遵循, 但几乎任何电生理学实验室都可以使用多重单细胞 rt-pcr 技术。

Introduction

大脑皮层包括多种细胞类型, 涉及各种生理过程。考虑到皮层细胞类型的形态学、生理和分子多样性, 它们的识别和表征是理解其特定功能的先决条件, 这是非常有挑战性的.1 ,2,3,4。

单细胞复合 rt-pcr 是基于膜片钳和 rt-pcr 技术相结合的。它可以同时探测30多个预定义基因在 electrophysiologically 识别细胞5中的表达。在记录吸管中加入神经元示踪剂进一步允许在组织化学揭示6、7、8、9后, 记录细胞的形态学特征,10. 基于对其表型性状5、9、10、11、12 的多元分析, 这是一种非常有用的神经类型分类方法. ,13,14。单细胞多重 rt-pcr 也适合于非神经细胞的特征, 如星形胶质细胞15,16,17, 几乎可以应用到每一个大脑结构18, 19、20、21、22、23和单元格类型, 假设它们可以在全单元格配置中进行记录。

这项技术是非常方便的识别细胞源和/或目标的传输系统7,8,15,16,20,21, 24,25,26,27,28, 特别是缺乏特定抗体时。它依赖于从视觉上识别出的细胞29的补丁钳录音, 因此也允许在特定的蜂窝环境8,15,16中定位细胞。此外, 由于大脑组织的细胞构筑被保存在脑切片中, 这种方法还可以研究具有神经元和非神经元元素的特征细胞的解剖关系7,8,18。

由于该技术受所收获细胞质数量和 RT 效率的限制, 在低拷贝数的情况下检测 mRNA 可能很难。虽然基于 RNaseq 技术的其他方法允许分析单个单元3、4、30、31的整个转录, 但它们需要高吞吐量的昂贵音序器不一定每个实验室都可以使用。由于单细胞复用 rt-pcr 技术采用终点 pcr 方法, 因此只需要广泛使用 thermocyclers。它可以很容易地开发在装有电生理设置的实验室, 不需要昂贵的设备。它可以在一天内提供一组预定义基因的定性分析。因此, 这种方法可以很容易地获得单个细胞的分子特征。

Protocol

所有使用动物的实验程序都严格按照法国的规定进行 (农村 R214/87 R214/130), 符合欧洲经济共同体 (86/609/欧洲经济共同体) 和法国国家宪章的道德准则。动物实验伦理学研究。所有议定书均经查尔斯·达尔文道德委员会批准, 并提交法国教育部和研究部 (批准 2015 061011367540)。IBPS 动物设施由法国当局 (A75-05-24) 认证。 1. 初步考虑 注: 为避免污染, 在进行单细胞 rt-pc…

Representative Results

图 3显示了多路 rt-pcr 的代表性验证。该协议旨在同时探索12种不同基因的表达。水泡谷氨酸转运 vGluT1 被作为一个积极的控制谷氨酸神经元42。以 GABA 合成酶 (GAD65 和 67)、神经肽 Y (NPY) 和生长抑素 (SOM) 为 GABAergic 中间神经元3、5、11的标记物。cyclooxygenase-2 酶 (COX-2)…

Discussion

单细胞多重 rt-pcr 后贴片钳可以同时可靠地探测30多个基因在 electrophysiologically 识别细胞5的表达。在单细胞水平上分析基因表达需要高效的 PCR 引物。最限制的步骤之一是单元格内容的集合。其效率取决于贴片吸管尖端的直径, 它必须尽可能大, 同时匹配的细胞大小。吸管与1-2 µm 开尖直径被证明是适合大多数神经元类型。同样重要的是要确保只收集细胞的内容, 而不是周围的组织?…

Offenlegungen

The authors have nothing to disclose.

Acknowledgements

我们感谢亚历山大 Mourot 博士对手稿的评论。这项工作得到了 la 研究所 (2011 MALZ 003 01) 赠款的支持;ANR-15-CE16-0010 和 ANR-17-CE37-0010-03), 大人物是支持的奖学金从研究所阿尔茨海默氏症。我们感谢 IBPS 的动物设施 (巴黎, 法国)。

Materials

MACAW v.2.0.5 NCBI Multiple alignement for primer design
Dithiothreitol VWR 443852A RT
Random primers Sigma-Aldrich (Merck) 11034731001 RT
dNTPs GE Healthcare Life Sciences 28-4065-52 RT and PCR
RNasin Ribonuclease Inhibitors Promega N2511 RT
SuperScript II Reverse Transcriptase Invitrogen 18064014 RT
Taq DNA Polymerase Qiagen 201205 PCR
Mineral Oil Sigma-Aldrich (Merck) M5904-5ML PCR
PCR primers Sigma-Aldrich (Merck) PCR / desalted and diluted at 200 µM
Tubes, 0.5 mL, flat cap ThermoFisher Scientific AB0350 RT and PCR
BT10 Series – 10 µL Filter Tip Neptune Scientific BT10 RT and PCR
BT20 Series – 20 µL Filter Tip Neptune Scientific BT20 RT and PCR
BT200 Series – 200 µL Filter Tip Neptune Scientific BT200 RT and PCR
BT1000 Series – 1000 µL Filter Tip Neptune Scientific BT1000.96 RT and PCR
DNA Thermal Cylcer Perkin Elmer Cetus PCR
Ethidium Bromide Sigma-Aldrich (Merck) E1510-10ML Agarose gel electrophoresis
Tris-Borate-EDTA buffer Sigma-Aldrich (Merck) T4415-1L Agarose gel electrophoresis
UltraPure Agarose Life Technologies 16500-500 Agarose gel electrophoresis
ΦX174 DNA-Hae III Digest NEB (New England BioLabs) N3026S Agarose gel electrophoresis
EDA 290 Kodak Agarose gel electrophoresis
Electrophoresis Power supply EPS 3500 Pharmacia Biotech Agarose gel electrophoresis
Midi Horizontal Elecrophoresis Unit Model SHU13 Sigma-Aldrich (Merck) Agarose gel electrophoresis
Smooth paper with satin appearance Fisherbrand 1748B Patch clamp internal solution
Potassium Hydroxyde Sigma-Aldrich (Merck) 60377 Patch clamp internal solution
Ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid Sigma-Aldrich (Merck) E3889 Patch clamp internal solution
HEPES Sigma-Aldrich (Merck) H4034 Patch clamp internal solution
Potassium D-gluconate Sigma-Aldrich (Merck) G4500 Patch clamp internal solution
Magnesium chloride solution Sigma-Aldrich (Merck) M1028 Patch clamp internal solution
5500 Vapor Pressure Osmometer Wescor Patch clamp internal solution
Biocytin Sigma-Aldrich (Merck) B4261 Patch clamp internal solution
Sucrose Sigma-Aldrich (Merck) S5016 Slice preparation
D-(+)-Glucose monohydrate Sigma-Aldrich (Merck) 49159 Slice preparation
Sodium chloride Sigma-Aldrich (Merck) S6191 Slice preparation
Potassium chloride Sigma-Aldrich (Merck) 60128 Slice preparation
Sodium bicarbonate Sigma-Aldrich (Merck) 31437-M Slice preparation
Sodium phosphate monobasic Sigma-Aldrich (Merck) S5011 Slice preparation
Magnesium chloride solution Sigma-Aldrich (Merck) 63069 Slice preparation
Calcium chloride solution Sigma-Aldrich (Merck) 21115 Slice preparation
Kynurenic acid Sigma-Aldrich (Merck) K3375 Slice preparation
Isoflurane Piramal Healthcare UK Slice preparation
VT 1000S Leica Biosystems 14047235613 Slice preparation
Hydrogen peroxide solution Sigma-Aldrich (Merck) H1009 Patch Clamp set-up cleaning
Thin Wall Glass Capillaries with filament World Precision Instruments TW150F-4 Patch Clamp
PP-83 Narishige Patch Clamp
Eppendorf Microloader Eppendorf 5242956003 Patch Clamp
BX51WI Upright microscope Olympus Patch Clamp
XC-ST70/CE CCD B/W VIDEO CAMERA Sony Patch Clamp
Axopatch 200B Amplifier Molecular Devices Patch Clamp
Digidata 1440 Molecular Devices Patch Clamp
pCLAMP 10 software suite Molecular Devices Patch Clamp
10 mL syringe Terumo SS-10ES Expelling
E Series with Straight Body (Holder) Phymep 64-0997 Expelling
Sodium phosphate dibasic Sigma-Aldrich (Merck) S7907 Histochemical revelation
Sodium phosphate monobasic Sigma-Aldrich (Merck) S8282 Histochemical revelation
Paraformaldehyde Sigma-Aldrich (Merck) P6148 Histochemical revelation
Triton X-100 Sigma-Aldrich (Merck) X100 Histochemical revelation
Gelatin from cold water fish skin Sigma-Aldrich (Merck) G7041 Histochemical revelation
Streptavidin, Alexa Fluor 488 conjugate ThermoFisher Scientific S11223 Histochemical revelation
24-well plate Greiner Bio-One 662160 Histochemical revelation
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Tags

Single CellMultiplexReverse TranscriptionPolymerase Chain ReactionPatch-clampNeurowissenschaftenNeuronal PopulationTransmission SystemsGene ExpressionAstrocytesAnimal ModelsBrain DissectionVibratome SectioningWhole-cell RecordingCytoplasm HarvestingRT-PCRGDNA Contamination

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Devienne, G., Le Gac, B., Piquet, J., Cauli, B. Single Cell Multiplex Reverse Transcription Polymerase Chain Reaction After Patch-clamp. J. Vis. Exp. (136), e57627, doi:10.3791/57627 (2018).
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