单细胞基因表达谱使用流式细胞仪和qPCR内部标准
Summary
We describe a method to sort single mammalian cells and to quantify the expression of up to 96 target genes of interest in each cell. This method includes the use of internal qPCR standards to enable the estimation of absolute transcript counts.
Abstract
Gene expression measurements from bulk populations of cells can obscure the considerable transcriptomic variation of individual cells within those populations. Single-cell gene expression measurements can help assess the role of noise in gene expression, identify correlations in the expression of pairs of genes, and reveal subpopulations of cells that respond differently to a stimulus. Here, we describe a procedure to measure the expression of up to 96 genes in single mammalian cells isolated from a population growing in tissue culture. Cells are sorted into lysis buffer by fluorescence-activated cell sorting (FACS), and the mRNA species of interest are reverse-transcribed and amplified. Gene expression is then measured using a microfluidic real-time PCR machine, which performs up to 96 qPCR assays on up to 96 samples at a time. We also describe the generation and use of PCR amplicon standards to enable the estimation of the absolute number of each transcript. Compared with other methods of measuring gene expression in single cells, this approach allows for the quantification of more distinct transcripts than RNA FISH at a lower cost than RNA-Seq.
Introduction
在群体中的单个细胞可显示成均匀的生理刺激物1，2，3，4大不相同响应。细胞群体中的遗传变异是该各种反应机制之一，但也有几个非遗传因素，可以增加的响应的变异性，即使是在细胞的克隆群。例如，单个蛋白质和其他重要的信号传导分子的水平可以在小区通过小区基础而变化，从而引起变化在下游基因表达谱。此外，可发生在转录物5,6可被限制在一个相对小的数目每爆裂7，8，9的转录物的短持续时间的脉冲串的基因活化。这样随机性在基因激活可以大大促进生物反应的变异，可以在微生物10和在哺乳动物细胞中1提供选择优势，2响应于生理刺激。由于变异的遗传和非遗传来源，响应于刺激的任何给定的细胞的基因表达图谱可以由从本体响应的测量获得的平均基因表达谱大大不同。确定哪个个体细胞显示变异性响应于刺激的程度，需要对单个细胞的分离技术，用于感兴趣转录子的表达水平的测量，并将所得的表达数据的计算分析。
有用于测定基因表达的单细胞，覆盖范围广，成本几种方法，成绩单的数量探测和定量的精度。例如，单细胞RNA测序提供转录的覆盖范围和量化数千不同转录为在个体细胞中最高度表达的基因的能力的广泛的深度;然而，用这样的测序深度相关的成本可能是昂贵，虽然成本的不断降低。相反， 在原位杂交（smRNA FISH）单分子的RNA的荧光提供转录为精确定量甚至低表达以每感兴趣基因以合理的成本的基因;然而，只有靶基因的小的数目可以在通过该方法给定的细胞来测定。定量基于PCR的检测，在本协议中所述，提供这些技术之间的中间地带。这些测定使用的微流体实时PCR机在高达96的细胞的时间量化到感兴趣96转录。虽然每一个上述的方法具有所需的硬件成本，任何个别qPCR分析的成本相对低。该协议适于从一个由一个微流体实时PCR机的生产（协议ADP 41，Fluidigm公司）建议。要启用基于PCR的方法每个成绩单的绝对数量的估计，我们已经扩大了协议，使利用可在多个实验中使用准备靶基因扩增的内部控制。
作为这种技术的一个例子，通过肿瘤抑制基因p53的MCF-7人乳腺癌细胞调节的基因的表达的定量描述11。将细胞挑战与诱导DNA双链断裂的化学试剂。以前的研究已经表明，p53的应答DNA双链断裂表现出在个体细胞异质性很大，无论是在p53水平12的术语和在不同的靶基因11的激活。此外，P53调控的超过100个的表达良好表征参与许多下游途径，包括细胞周期停滞，细胞凋亡和衰老13,14靶基因。因为在每个小区中的p53介导的应答是既复杂又变量的系统的好处，从一种方法的分析中，几乎100靶基因可同时在单个细胞，如以下所述进行探测。稍作修改（如单细胞分离和裂解替代方法），该协议可以很容易地适合于研究范围广泛的哺乳动物细胞类型，转录物和细胞应答的。
通过适当的预先准备，圆细胞分类和基因表达测量的，可根据这个协议在一段三天进行。下面的时序建议：提前，选择感兴趣的成绩单，识别和验证那些transcr扩增cDNA的引物对IPTS，并准备标准，并利用这些引物引物混合物。在第1天，以下细胞治疗，收获和细胞进行排序，执行反转录和靶特异性扩增，并用外切核酸酶处理的样品以除去未掺入的引物。第2天，请对使用qPCR排序细胞质量控制。最后，在第3天，测量在使用微流体的qPCR的分选的细胞的基因表达。 图1总结了所涉及的步骤。
Protocol
Representative Results
Discussion
我们已经提出，用于从培养物中生长贴壁细胞群中分离个体哺乳动物细胞和用于测定在每个小区中的大约96个基因的表达的方法。良好的事前准备是至关重要的，此方法才能正常工作。特别是，具体到感兴趣的转录（步骤1.2-1.3）设计和测试的引物对是费时，但重要的步骤，因为引物确定单细胞测量的质量。一旦可靠的引物对已经获得，它们被用来从感兴趣的转录扩增的cDNA;扩增子然后在等摩尔量?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
我们要感谢V.卡普尔在CCR ETIB流式细胞仪的核心，她在这个协议的开发过程中进行细胞分选援。我们也感谢M. Raffeld和CCR LP分子诊断单位和朱军和NHLBI DNA测序与基因组学核心为他们在这个协议的开发过程中进行定量PCR援助。这项研究是由美国国立卫生研究院的校内计划的支持。
Materials
| RNeasy Plus Mini Kit | Qiagen | 74134 | |
| High Capacity cDNA Reverse Transcription Kit with RNase Inhibitor | ThermoFisher | 4374966 | |
| Phusion High-Fidelity DNA Polymerase | New England BioLabs | M0530S | |
| QIAquick Gel Extraction Kit | Qiagen | 28704 | |
| Quant-iT High-Sensitivity dsDNA Assay Kit | ThermoFisher | Q33120 | |
| 2.0-mL low adhesion microcentrifuge tubes | USA Scientific | 1420-2600 | |
| DNA Suspension Buffer | Teknova | T0221 | |
| Axygen 0.2-mL Maxymum Recovery Thin Wall PCR Tubes | Corning | PCR-02-L-C | |
| GE 96.96 Dynamic Array DNA Binding Dye Sample & Assay Loading Reagent Kit | Fluidigm | 100-3415 | |
| HyClone RPMI 1640 media | GE Healthcare Life Sciences | SH30027.01 | |
| Fetal Bovine Serum, Certified (US) | ThermoFisher | 16000-044 | |
| Antibiotic-Antimycotic Solution | Corning | 30-004-CI | |
| Neocarzinostatin | Sigma | N9162 | |
| ELIMINase | Decon Labs | 1101 | |
| SUPERase-In | ThermoFisher | AM2696 | |
| CellsDirect One-Step qRT-PCR Kit | ThermoFisher | 11753500 | |
| E. coli DNA | Affymetrix | 14380 10 MG | |
| ThermalSeal Sealing Film, Sterile | Excel Scientific | STR-THER-PLT | |
| BD FACSAria IIu | BD Biosciences | ||
| HyClone Trypsin 0.05% | GE Healthcare Life Sciences | SH30236.01 | |
| PBS, 1x | Corning | 21-040-CV | |
| Falcon 40µm Cell Strainer | Corning | 352340 | |
| Exonuclease I | New England BioLabs | M0293S | |
| SsoFast EvaGreen Supermix with Low ROX | Bio-Rad | 172-5210 | |
| 96.96 Dynamic Array IFC for Gene Expression (microfluidic qPCR chip) | Fluidigm | BMK-M-96.96 | |
| IFC Controller HX (loading machine) | Fluidigm | ||
| BioMark or BioMark HD (microfluidic qPCR machine) | Fluidigm | ||
| Real-Time PCR Analysis software | Fluidigm | ||
| MATLAB software | MathWorks |
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