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。

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The authors have nothing to disclose.

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结合在一起，使标准（步骤1.4）。这个步骤是重要的，因为在标准来估计绝对转录计数必需的;标准应进行一次，等分，并用于所有随后的几轮细胞分类和基因表达的测量。同样地，在天的细胞进行排序，这是特别importan吨至稀释使得裂解液的板（步骤3.7和3.11）时，小心使用低结合枪头和离心管的标准。针对细胞分选（步骤4.2）是另一个关键的一步;这必须以非常仔细地进行为细胞成功击中的9微升裂解缓冲液的靶在PCR板。 有几种修改，可以作出以使其适应各种研究需要的协议。这里，我们专注于一个DNA结合染料基的qPCR方法，它提供了用于定量基因表达的一个比较实惠的方法。然而，该方法具有用于产生高背景信号，因为任何扩增的DNA，即使是脱靶序列，结果在荧光信号的电位。这样的背景下可以通过确保用于扩增特定的目标的引物对产生具有单峰和正确大小的单一的PCR产物（ 图2的熔融曲线被最小化）。如果背景是仍然使用这种质量控制方法后一个问题，基于探针定量PCR方法可以在试剂的更大的成本可以用来最大限度地减少脱靶检测，虽然。另一种选择是嵌套PCR方法中，其中一组引物在RTSTA步骤用于反向转录和扩增转录物的大区域和第二组引物，其放大包含在该大区域中的较小区域中，使用通过qPCR来测量基因表达。这种方法已被证实可以改善DNA结合基于染料的qPCR 23的特异性。 有几种方法可用于分离单个细胞的基因表达分析。细胞分离方法的选择取决于几个因素，包括设备（诸如荧光激活细胞分选）的可用性，该细胞的来源，以进行评估（如建立的细胞系对初级细胞从组织），或与该细胞必须被隔离的速度。在这个协议中提出的例子中，我们使用表达清楚地检测到荧光标记蛋白的细胞系的FACS。 FACS拥有罕见的细胞检测能力和较快的细胞分离的好处。该方法的一个挑战是，感兴趣的细胞的沉积到裂解缓冲液在96孔PCR板中的所需体积小可以是低效的，并且可能需要在细胞分选几何的优化，以确保成功的隔离。可以在较低的速度和/或较高的成本导致细胞分离更大的精度的各种途径包括单个细胞的micropipetting，从肿瘤样本特定细胞的激光捕获显微切割，或使用微流体系统的（ 例如，Fluidigm公司C1系统）。 这里所描述的协议中的一个主要的好处是，内部控制，系列稀释纯化的PCR扩增子，例如<html> nable在每个小区的每个成绩单的绝对数量的估计。没有这些标准，可以基于从（C T）值导出的计算来获得的基因表达的唯一相对水平。然而，这些标准，绝对转录计数可以被估计，最好是通过精确定量扩增子的标准（ 图8）的范围内的内插。作为基因表达的定量的任何基于PCR的方法中，精确绝对定量要求每个过程的效率，包括反转录的知识。 在单细胞定量转录水平的电流挑战是检测的许多方法的极限估计为每个细胞24 10-100的mRNA，从而防止转录的显著百分比的可靠定量。作为一个替代方法，可以使用smRNA FISH量化特别感兴趣的目标屁股="外部参照"&gt; 25，26日 ，包括那些极少数的成绩单。在smRNA FISH进步已经扩大不同转录物可在一次27来检测，给予适当的设备的数量和可在一次28异型细胞的数目。虽然smRNA FISH有它自己的限制（潜在假阳性和假阴性转录检测，限制每个细胞只有少数靶基因，设备和试剂的成本），它可以提供一种有效的方法，以补充和验证从结果感兴趣的靶基因的一个子集的基于定量PCR分析。

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。

<table border="0" cellpadding="0" cellspacing="0" width="988">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:488px;">RNeasy Plus Mini Kit</td>
			<td style="width:187px;">Qiagen</td>
			<td style="width:147px;">74134</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">High Capacity cDNA Reverse Transcription Kit with RNase Inhibitor</td>
			<td>ThermoFisher</td>
			<td>4374966</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Phusion High-Fidelity DNA Polymerase</td>
			<td>New England BioLabs</td>
			<td>M0530S</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">QIAquick Gel Extraction Kit</td>
			<td>Qiagen</td>
			<td>28704</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Quant-iT High-Sensitivity dsDNA Assay Kit</td>
			<td>ThermoFisher</td>
			<td>Q33120&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">2.0-mL low adhesion microcentrifuge tubes</td>
			<td>USA Scientific</td>
			<td>1420-2600</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DNA Suspension Buffer</td>
			<td>Teknova</td>
			<td>T0221</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Axygen 0.2-mL Maxymum Recovery Thin Wall PCR Tubes</td>
			<td>Corning</td>
			<td>PCR-02-L-C</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">GE 96.96 Dynamic Array DNA Binding Dye Sample &#38; Assay Loading Reagent Kit</td>
			<td>Fluidigm</td>
			<td>100-3415</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HyClone RPMI 1640 media</td>
			<td>GE Healthcare Life Sciences</td>
			<td>SH30027.01</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fetal Bovine Serum, Certified (US)</td>
			<td>ThermoFisher</td>
			<td>16000-044</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Antibiotic-Antimycotic Solution</td>
			<td>Corning</td>
			<td>30-004-CI</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Neocarzinostatin</td>
			<td>Sigma</td>
			<td>N9162</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">ELIMINase</td>
			<td>Decon Labs</td>
			<td>1101</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">SUPERase-In</td>
			<td>ThermoFisher</td>
			<td>AM2696</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CellsDirect One-Step qRT-PCR Kit</td>
			<td>ThermoFisher</td>
			<td>11753500</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">E. coli DNA</td>
			<td>Affymetrix</td>
			<td>14380 10 MG</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">ThermalSeal Sealing Film, Sterile</td>
			<td>Excel Scientific</td>
			<td>STR-THER-PLT</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">BD FACSAria IIu</td>
			<td>BD Biosciences</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HyClone Trypsin 0.05%</td>
			<td>GE Healthcare Life Sciences</td>
			<td>SH30236.01</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PBS, 1x</td>
			<td>Corning</td>
			<td>21-040-CV</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Falcon 40&#181;m Cell Strainer</td>
			<td>Corning</td>
			<td>352340</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Exonuclease I</td>
			<td>New England BioLabs</td>
			<td>M0293S</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">SsoFast EvaGreen Supermix with Low ROX</td>
			<td>Bio-Rad</td>
			<td>172-5210</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">96.96 Dynamic Array IFC for Gene Expression (microfluidic qPCR chip)</td>
			<td>Fluidigm</td>
			<td>BMK-M-96.96</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">IFC Controller HX (loading machine)</td>
			<td>Fluidigm</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">BioMark or BioMark HD (microfluidic qPCR machine)</td>
			<td>Fluidigm</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Real-Time PCR Analysis software&#160;</td>
			<td>Fluidigm</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MATLAB software</td>
			<td>MathWorks</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

single-cell gene expression profiling using facs and qpcr with internal standards

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.

该协议的一般概述示于图1，包括用于细胞治疗，单细胞通过FACS中，分离步骤产生和从单细胞裂解物，单细胞cDNA文库中的确认cDNA文库预扩增排序井，和基因表达通过qPCR测量。 在单细胞的分离和基因表达分析的准备，有必要首先确定每个感兴趣的靶基因有效的寡核苷酸引物对。 图2示出的引物的质量控制方法的两个例子。熔融曲线生成与引物对被测试以下的qPCR。有效的引物导致具有单峰（ 图2A，绿色曲线）熔体曲线。如果多个峰（ 图2A，蓝色曲线）或具有明显的肩（一个曲线FIGURê2A，红色曲线）都存在，这表明的多重PCR产物的存在，以及引物，应重新设计。作为第二质量控制方法，琼脂糖凝胶电泳可用于在视觉上确认正确大小的单一条带的存在对于每个引物对进行测试（ 图2B）。多个波段或错误的大小的条带表明引物是无效的（ 图2B）。 以下引物验证和从正确的扩增子的qPCR标准库的产生，可进行细胞分选。在图3的示例排序方案被表明。每个排序板应包括含有稀释系列的扩增子的标准孔中，每个靶扩增子的10，100，1000，或10,000拷贝。此外，还建议以包括在其中没有细胞，10个细胞，或100个细胞进行排序，除了单细胞井孔中。由于分选效率，经常需要确定在哪些单电池已被成功地沉积在板的孔中。对于此验证步骤，以下为逆转录和特定靶序列扩增的方法，定量PCR应该执行测量持家基因（ 图4）的表达。对于扩增子的标准孔中（ 图4，黑色曲线）应显示均匀隔开的扩增曲线。还应该在对应于"无细胞"井曲线清楚地分离（ 图4;红色曲线），"10-细胞"孔中（ 图4;蓝色曲线），和"100单元"孔中（ 图4 ;紫色曲线）。明确分开也应发生相应于成功的细胞沉积（ 图4中的"1单元"井;用（C T）值低于那些无细胞控制，但G绿色扩增曲线与邻近者为无细胞对照（C T）的值）绿色的曲线;比为10细胞对照）与不成功细胞沉积（ 图4 reater。一旦与单个细胞裂解物的井已经确定，从孔的cDNA（可能来自多个板）可以被转移到微流体的qPCR芯片，每个井一单细胞裂解物。例如， 图5示出了一个假设阵列，从单个细胞从三个不同的排序的96孔板成用于定量PCR分析的单个新板结合的cDNA。 来分析微流体的qPCR结果，即有可能没有收到细胞样本应首先被除去，由许多缺失或零基因表达测量（ 图7中由箭头指示的行）或异常低的持家基因的表达所指示的。对于每个测定，具有熔体峰任何测量不匹配的那些水净化的在相同的测定r个采样也应除去。除去坏样品和测量之后，线性回归可以在对应于扩增子的标准测量来进行估计的每个单细胞样品中每个感兴趣的转录物的绝对计数。 图8示出了这些基因表达的估计，在分子×逆转录效率的单位测量，如beeswarm和小提琴地块的可视化。在本例中，未经处理的细胞显示了广泛CDKN1A表达水平，以在所有的不表达CDKN1A许多细胞，并与他人表达在跨越两个数量级水平的基因。用新制癌菌素（NCS）处理3小时后，大部分细胞表达CDKN1A在更高水平，可变性降低到幅度大约一个订单。作为治疗的增加的持续时间，CDKN1A表达的平均水平降低，并且在变性<html>表达扩大。 <img alt="图1" src="/files/ftp_upload/55219/55219fig1.jpg" /> 图 1: 在单细胞基因表达分析的步骤概述。处理的收获细胞并通过FACS分选的直接进入裂解缓冲液。感兴趣的mRNA种类被逆转录，并将所得的cDNA通过PCR扩增。每个样品中持家基因表达通过定量PCR测量，以确定哪些样本实际接收的小区。在通过此质量控制测试样品，高达96个基因的表达是使用微流体的qPCR测量。这个数字已经从以前的出版物11修改。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55219/55219fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 19 / 55219fig2.jpg"/&gt; 图2: 引物的检测的实施例结果。 （a）熔体从定量PCR曲线。蓝色熔解曲线（TNFRSF10D）具有多个峰和红色熔解曲线（TRIM22）具有"肩"，它相当于第二峰值靠近第一峰。这些表明，在这些反应中使用的引物扩增的多个目标和需要重新设计。绿色熔解曲线（SCN3B）具有单峰，与该放大单个目标的引物是一致的。 （B）放大了琼脂糖凝胶DNA运行。对于SCN3B，TRIM22和TRPM2车道包含多个频段;用于制造的那些DNA扩增子的引物扩增的多个目标和需要重新设计。对于TNFRSF10D车道具有单一条带，与扩增单个目标的引物是一致的。值得注意的是，对于SCN3B引物通过熔融曲线测试但没有通过凝胶测试，而那些对TNFRSF10D通过凝胶测试，但失败的熔解曲线测试;底漆验证这两种方法是互补的。在该例子中，人们可以得出结论，所有四个引物组需要重新设计。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55219/55219fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/55219/55219fig3.jpg" /> 图3:板材地图细胞分选。该板地图包括1-细胞孔（红色），10和100细胞的孔（深红色），标准（绿），和无细胞孔（白色）。包括在每个板的每个标准的两个重复有助于补偿在标准变异性。包括10个单元和100个细胞孔作为一个健全性检查基因表达有帮助。无细胞孔充当阴性对照。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55219/55219fig3large.jpg" target="_blank">请点击此处查看T的放大版本。</a>他的身影。 <img alt="图4" src="/files/ftp_upload/55219/55219fig4.jpg" /> 图4:从某种质量控制测量GAPDH表达定量PCR扩增曲线的例子。无细胞样品（红）显示表达的背景水平。 1-细胞样品（绿色）分为两个不同的组，一组具有高表达和一个具有类似于无细胞样品低表达。高表达的样本中排序最有可能获得一个真正的细胞;低表达的样本很可能没有。 10细胞（蓝色）和100细胞（紫色）的样品显示出比1细胞样品更高的表达; 10细胞样本接近，因为那种低效率的表达最高的1 - 细胞样本。标准（黑色）是均匀分布的，如预期。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55219/55219fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> &lt;/ P&gt; <img alt="图5" src="/files/ftp_upload/55219/55219fig5.jpg" /> 图5:示例的实施例混合曲目板图。该板地图包括来自三个不同的板（行AG）1-细胞样品，来自所有三个板（H1-H8），无细胞对照（H9-H10），混合，标准和无模板对照为定量PCR（H11-H12 ）。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55219/55219fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图6" src="/files/ftp_upload/55219/55219fig6.jpg" /> 图6: 微流体的qPCR芯片装载（1，2，3，...）的顺序的图。凹口（A1）为在板的左上角。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55219/55219fig6large.jpg" target="_blank">请点击此处查看THI一个更大的版本</a>的身影。 <img alt="图7" src="/files/ftp_upload/55219/55219fig7.jpg" /> 图 7: 定量PCR结果的热图。每一行代表一个1 - 细胞样本，标准或控制;每一列表示一个测量的转录。用黑色方块或X（箭头）的一个不寻常的行数可能表明，没有收到实际的细胞样本。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55219/55219fig7large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图8" src="/files/ftp_upload/55219/55219fig8.jpg" /> 图8:Beeswarm和小提琴地块。在一个beeswarm图（蓝色点），每个点代表在一个特定的细胞的基因表达。在一个小提琴曲线（灰色形状）中，"小提琴"的宽度代表基因表达measu的频率围绕在分析细胞群的特定水平rements。淡蓝色的条代表的最低标准。上面的蓝色条测量标准之间的插值，而蓝色酒吧内测量推断。这里显示的数据是在表达与新制癌菌素（NCS）处理所示时间金星标记p53在为了诱导DNA双链断裂MCF-7细胞CDKN1A表达的测量。这个数字已经从以前的出版物11修改。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55219/55219fig8large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 条件 </td><td> 温度 </td><td> 时间 </td></tr><tr><td>保持</td><td> 50℃ </td><td> 15分钟</td></tr><tr><td>保持</td><td> 95℃ </td><td> 2分钟</td></tr><tr><td rowspan="2"> 20次</td><td> 95℃ </td><td> 15秒</td></tr><tr><td> 60℃ </td><td> 4分钟</td></tr><tr><td>保持</td><td> 4℃ </td><td> ∞ </td></tr></tbody></table> 表1:反转录和特定目标扩增（RTSTA）热循环程序。 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 条件 </td><td> 温度 </td><td> 时间 </td></tr><tr><td>保持</td><td> 37℃ </td><td> 30分钟</td></tr><tr><td>保持</td><td> 80℃ </td><td> 15分钟</td></tr><tr><td>保持</td><td> 4℃ </td><td> ∞ </td></tr></tbody></table>表2:核酸外切酶I治疗（EXOI）热循环程序。 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 零件 </td><td> 音量/孔（微升） </td><td> 96卷瓦特/ 10％的超出（微升） </td></tr><tr><td> 2X反应混合物</td><td> 5.00 </td><td> 528.00 </td></tr><tr><td>逆转录/聚合酶混合物</td><td> 0.20 </td><td> 21.12 </td></tr><tr><td> 10X STA引物混合物</td><td> 1.00 </td><td> 105.60 </td></tr><tr><td> 2.64 U /μL（稀）RNA酶抑制剂</td><td> 0.02 </td><td> 2.00 </td></tr><tr><td>不含核酸酶的水</td><td> 0.78 </td><td> 82.48 </td></tr><tr><td> 总 </td><td> 7.00 </td><td> 739.20 </td></tr></tbody></table>表3:细胞裂解缓冲液的组成。 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 零件 </td><td> 音量/孔（微升） </td><td> 96卷瓦特/ 10％的超出（微升） </td></tr><tr><td>不含核酸酶的水</td><td> 2.52 </td><td> 266.00 </td></tr><tr><td>核酸外切酶I反应缓冲液（10×） </td><td> 0.36 </td><td> 38.00 </td></tr><tr><td>核酸外切酶I（20U /微升） </td><td> 0.72 </td><td> 76.00 </td></tr><tr><td> 总 </td><td> 3.60 </td><td> 380.00 </td></tr></tbody></table> 表4:外切核酸酶的组分混合来对待扩增的cDNA样品。

Watch this Scientific Journal Video about Single-cell Gene Expression Profiling Using FACS and qPCR with Internal Standards at JoVE.com

使用 FACS 和 qPCR 及内标进行单细胞基因表达分析 |遗传学 |视频

Single-cell Gene Expression Profiling Using FACS and qPCR with Internal Standards

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.

单细胞基因表达谱使用流式细胞仪和qPCR内部标准

Gene expression measurements from bulk populations of cells can obscure the considerable transcriptomic variation of individual cells within those ...

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16.3K 次观看.  National Cancer Institute. 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.

视频: Single-cell Gene Expression Profiling Using FACS and qPCR with Internal Standards

Parallel Measurement of Circadian Clock Gene Expression and Hormone Secretion in Human Primary Cell Cultures

Circadian clocks are functional in all light-sensitive organisms, allowing for an adaptation to the external world by anticipating daily environmental changes. Considerable progress in our understanding of the tight connection between the circadian clock and most aspects of physiology has been made in the field over the last decade. However, unraveling the molecular basis that underlies the function of the circadian oscillator in humans stays of highest technical challenge. Here, we provide a detailed description of an experimental approach for long-term (2-5 days) bioluminescence recording and outflow medium collection in cultured human primary cells. For this purpose, we have transduced primary cells with a lentiviral luciferase reporter that is under control of a core clock gene promoter, which allows for the parallel assessment of hormone secretion and circadian bioluminescence. Furthermore, we describe the conditions for disrupting the circadian clock in primary human cells by transfecting siRNA targeting CLOCK. Our results on the circadian regulation of insulin secretion by human pancreatic islets, and myokine secretion by human skeletal muscle cells, are presented here to illustrate the application of this methodology. These settings can be used to study the molecular makeup of human peripheral clocks and to analyze their functional impact on primary cells under physiological or pathophysiological conditions.

Here, we describe settings to monitor in parallel circadian bioluminescence and the secretory activity of human islet cells and primary myotubes. For this, we employed lentiviral gene delivery of a luciferase core clock reporter, followed by in vitro synchronization and collection of outflow medium by continuous cell perifusion.

在人原代细胞培养生物钟基因表达及荷尔蒙分泌的并行测量

Circadian clocks are functional in all light-sensitive organisms, allowing for an adaptation to the external world by anticipating daily environmental ...

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遗传学

Perturbations of Circulating miRNAs in Irritable Bowel Syndrome Detected Using a Multiplexed High-throughput Gene Expression Platform

The gene expression platform assay allows for robust and highly reproducible quantification of the expression of up to 800 transcripts (mRNA or miRNAs) in a single reaction. The miRNA assay counts transcripts by directly imaging and digitally counting miRNA molecules that are labeled with color-coded fluorescent barcoded probe sets (a reporter probe and a capture probe). Barcodes are hybridized directly to mature miRNAs that have been elongated by ligating a unique oligonucleotide tag (miRtag) to the 3&#39; end. Reverse transcription and amplification of the transcripts are not required. Reporter probes contain a sequence of six color positions populated using a combination of four fluorescent colors. The four colors over six positions are used to construct a gene-specific color barcode sequence. Post-hybridization processing is automated on a robotic prep station. After hybridization, the excess probes are washed away, and the tripartite structures (capture probe-miRNA-reporter probe) are fixed to a streptavidin-coated slide via the biotin-labeled capture probe. Imaging and barcode counting is done using a digital analyzer. The immobilized barcoded miRNAs are visualized and imaged using a microscope and camera, and the unique barcodes are decoded and counted. Data quality control (QC), normalization, and analysis are facilitated by a custom-designed data processing and analysis software that accompanies the assay software. The assay demonstrates high linearity over a broad range of expression, as well as high sensitivity. Sample and assay preparation does not involve enzymatic reactions, reverse transcription, or amplification; has few steps; and is largely automated, reducing investigator effects and resulting in high consistency and technical reproducibility. Here, we describe the application of this technology to identifying circulating miRNA perturbations in irritable bowel syndrome.

We describe the use of a multiplexed high-throughput gene expression platform that quantitates gene expression by barcoding and counting molecules in biological substrates without the need for amplification. We used the platform to quantitate microRNA (miRNA) expression in whole blood in subjects with and without irritable bowel syndrome.

肠易激综合征的循环miRNA的扰动检测使用复用高通量基因表达平台

The gene expression platform assay allows for robust and highly reproducible quantification of the expression of up to 800 transcripts (mRNA or miRNAs) in ...

Single-cell Gene Expression Using Multiplex RT-qPCR to Characterize Heterogeneity of Rare Lymphoid Populations

Gene expression heterogeneity is an interesting feature to investigate in lymphoid populations. Gene expression in these cells varies during cell activation, stress, or stimulation. Single-cell multiplex gene expression enables the simultaneous assessment of tens of genes1,2,3. At the single-cell level, multiplex gene expression determines population heterogeneity4,5. It allows for the distinction of population heterogeneity by determining both the probable mix of diverse precursor stages among mature cells and also the diversity of cell responses to stimuli.
Innate lymphoid cells (ILC) have been recently described as a population of innate effectors of the immune response6,7. In this protocol, cell heterogeneity of the ILC hepatic compartment is investigated during homeostasis.
Currently, the most widely used technique to assess gene expression is RT-qPCR. This method measures gene expression only one gene at a time. Additionally, this method cannot estimate heterogeneity of gene expression, since multiple cells are needed for one test. This leads to the measurement of the average gene expression of the population. When assessing large numbers of genes, RT-qPCR becomes a time-, reagent-, and sample-consuming method. Hence, the trade-offs limit the number of genes or cell populations that can be evaluated, increasing the risk of missing the global picture.
This manuscript describes how single-cell multiplex RT-qPCR can be used to overcome these limitations. This technique has benefited from recent microfluidics technological advances1,2. Reactions occurring in multiplex RT-qPCR chips do not exceed the nanoliter-level. Hence, single-cell gene expression, as well as simultaneous multiple gene expression, can be performed in a reagent-, sample-, and cost-effective manner. It is possible to test cell gene signature heterogeneity at the clonal level between cell subsets within a population at different developmental stages or under different conditions4,5. Working on rare populations with large numbers of conditions at the single-cell level is no longer a restriction.

This protocol describes how to assess the expression of a large array of genes at the clonal level. Single-cell RT-qPCR produces highly reliable results with a strong sensitivity for hundreds of samples and genes.

单细胞基因表达利用多重RT-qPCR的表征罕见的淋巴人群的异质性

Gene expression heterogeneity is an interesting feature to investigate in lymphoid populations. Gene expression in these cells varies during cell ...

Detection of Copy Number Alterations Using Single Cell Sequencing

Detection of genomic changes at single cell resolution is important for characterizing genetic heterogeneity and evolution in normal tissues, cancers, and microbial populations. Traditional methods for assessing genetic heterogeneity have been limited by low resolution, low sensitivity, and/or low specificity. Single cell sequencing has emerged as a powerful tool for detecting genetic heterogeneity with high resolution, high sensitivity and, when appropriately analyzed, high specificity. Here we provide a protocol for the isolation, whole genome amplification, sequencing, and analysis of single cells. Our approach allows for the reliable identification of megabase-scale copy number variants in single cells. However, aspects of this protocol can also be applied to investigate other types of genetic alterations in single cells.

Single cell sequencing is an increasingly popular and accessible tool for addressing genomic changes at high resolution. We provide a protocol that uses single cell sequencing to identify copy number alterations in single cells.

单细胞测序拷贝数改变的检测

Detection of genomic changes at single cell resolution is important for characterizing genetic heterogeneity and evolution in normal tissues, cancers, and ...

Detection of microRNA Expression in Peritoneal Membrane of Rats Using Quantitative Real-time PCR

MicroRNAs (miRNAs) are small noncoding RNAs that regulate messenger RNA expression post-transcriptionally. The miRNA expression profile has been investigated in various organs and tissues in rat. However, standard methods for the purification of miRNAs and detection of their expression in rat peritoneal membrane have not been well established. We have developed an effective and reliable method to purify and quantify miRNAs using quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR) in rat peritoneal membrane. This protocol consists of four steps: 1) purification of peritoneal membrane sample; 2) purification of total RNA including miRNA from peritoneal membrane sample; 3) reverse transcription of miRNA to produce cDNA; and 4) qRT-PCR to detect miRNA expression. Using this protocol, we successfully determined that the expression of six miRNAs (miRNA-142-3p, miRNA-21-5p, miRNA-221-3p, miRNA-223-3p, miRNA-327, and miRNA-34a-5p) increased significantly in the peritoneal membrane of a rat peritoneal fibrosis model compared with those in control groups. This protocol can be used to study the profile of miRNA expression in the peritoneal membrane of rats in many pathological conditions.

Here we present a protocol for the detection of microRNA expression in rat peritoneal membrane using quantitative real-time reverse-transcription polymerase chain reaction. This method is suitable for studying the microRNA expression profile in rat peritoneal membrane in several pathological conditions.

使用定量实时PCR检测大鼠腹膜中的微小RNA表达

MicroRNAs (miRNAs) are small noncoding RNAs that regulate messenger RNA expression post-transcriptionally. The miRNA expression profile has been ...

Studying Cell Cycle-regulated Gene Expression by Two Complementary Cell Synchronization Protocols

The gene expression program of the cell cycle represents a critical step for understanding cell cycle-dependent processes and their role in diseases such as cancer. Cell cycle-regulated gene expression analysis depends on cell synchronization into specific phases. Here we describe a method utilizing two complementary synchronization protocols that is commonly used for studying periodic variation of gene expression during the cell cycle. Both procedures are based on transiently blocking the cell cycle in one defined point. The synchronization protocol by hydroxyurea (HU) treatment leads to cellular arrest in late G1/early S phase, and release from HU-mediated arrest provides a cellular population uniformly progressing through S and G2/M. The synchronization protocol by thymidine and nocodazole (Thy-Noc) treatment blocks cells in early mitosis, and release from Thy-Noc mediated arrest provides a synchronized cellular population suitable for G1 phase and S phase-entry studies. Application of both procedures requires monitoring of the cell cycle distribution profiles, which is typically performed after propidium iodide (PI) staining of the cells and flow cytometry-mediated analysis of DNA content. We show that the combined use of two synchronization protocols is a robust approach to clearly determine the transcriptional profiles of genes that are differentially regulated in the cell cycle (i.e. E2F1 and E2F7), and consequently to have a better understanding of their role in cell cycle processes. Furthermore, we show that this approach is useful for the study of mechanisms underlying drug-based therapies (i.e. mitomycin C, an anticancer agent), because it allows to discriminate genes that are responsive to the genotoxic agent from those solely affected by cell cycle perturbations imposed by the agent.

We report two cell synchronization protocols that provide a context for studying events related to specific phases of the cell cycle. We show that this approach is useful for analyzing the regulation of specific genes in an unperturbed cell cycle or upon exposure to agents affecting the cell cycle.

通过两个互补细胞同步方案研究细胞周期调控的基因表达

The gene expression program of the cell cycle represents a critical step for understanding cell cycle-dependent processes and their role in diseases such ...

Sample Preparation and Analysis of RNASeq-based Gene Expression Data from Zebrafish

The analysis of global gene expression changes is a valuable tool for identifying novel pathways underlying observed phenotypes. The zebrafish is an excellent model for rapid assessment of whole transcriptome from whole animal or individual cell populations due to the ease of isolation of RNA from large numbers of animals. Here a protocol for global gene expression analysis in zebrafish embryos using RNA sequencing (RNASeq) is presented. We describe preparation of RNA from whole embryos or from cell populations obtained using cell sorting in transgenic animals. We also describe an approach for analysis of RNASeq data to identify enriched pathways and Gene Ontology (GO) terms in global gene expression data sets. Finally, we provide a protocol for validation of gene expression changes using quantitative reverse transcriptase PCR (qRT-PCR). These protocols can be used for comparative analysis of control and experimental sets of zebrafish to identify novel gene expression changes, and provide molecular insight into phenotypes of interest.

This protocol presents an approach for whole transcriptome analysis from zebrafish embryos, larvae, or sorted cells. We include isolation of RNA, pathway analysis of RNASeq data, and qRT-PCR-based validation of gene expression changes.

斑马鱼 RNASeq-based 基因表达数据的样品制备与分析

The analysis of global gene expression changes is a valuable tool for identifying novel pathways underlying observed phenotypes. The zebrafish is an ...

Generation of a Gene-disrupted Streptococcus mutans Strain Without Gene Cloning

Typical methods for the elucidation of the function of a particular gene involve comparative phenotypic analyses of the wild-type strain and a strain in which the gene of interest has been disrupted. A gene-disruption DNA construct containing a suitable antibiotic resistance marker gene is useful for the generation of gene-disrupted strains in bacteria. However, conventional construction methods, which require gene cloning steps, involve complex and time-consuming protocols. Here, a relatively facile, rapid, and cost-effective method for targeted gene disruption in Streptococcus mutans is described. The method utilizes a 2-step fusion polymerase chain reaction (PCR) to generate the disruption construct and electroporation for genetic transformation. This method does not require an enzymatic reaction, other than PCR, and additionally offers greater flexibility in terms of the design of the disruption construct. Employment of electroporation facilitates the preparation of competent cells and improves the transformation efficiency. The present method may be adapted for the generation of gene-disrupted strains of various species.

Generation of a Gene-disrupted Streptococcus mutans Strain Without Gene Cloning

We describe a facile, rapid, and relatively inexpensive method for the generation of gene-disrupted Streptococcus mutans strains; this technique may be adapted for the generation of gene-disrupted strains of various species.

无基因克隆的基因破坏的变形链球菌菌株的生成

Typical methods for the elucidation of the function of a particular gene involve comparative phenotypic analyses of the wild-type strain and a strain in ...

Measuring Gene Expression in Bombarded Barley Aleurone Layers with Increased Throughput

The aleurone layer of barley grains is an important model system for hormone-regulated gene expression in plants. In aleurone cells, genes required for germination or early seedling development are activated by gibberellin (GA), while genes associated with stress responses are activated by abscisic acid (ABA). The mechanisms of GA and ABA signaling can be interrogated by introducing reporter gene constructs into aleurone cells via particle bombardment, with the resulting transient expression measured using enzyme assays. An improved protocol is reported that partially automates and streamlines the grain homogenization step and the enzyme assays, allowing significantly more throughput than existing methods. Homogenization of the grain samples is carried out using an automated tissue homogenizer, and GUS (&#946;-glucuronidase) assays are carried out using a 96-well plate system. Representative results using the protocol suggest that phospholipase D activity may play an important role in the activation of HVA1 gene expression by ABA, through the transcription factor TaABF1.

An improved protocol is presented for the measurement of transient gene expression from reporter constructs in barley aleurone cells after particle bombardment. The combination of automated grain grinding with 96-well plate enzyme assays provides high throughput for the procedure.

提高吞吐量的轰击大麦糊粉层层中基因表达的测定

The aleurone layer of barley grains is an important model system for hormone-regulated gene expression in plants. In aleurone cells, genes required for ...

Metabolic Labeling and Profiling of Transfer RNAs Using Macroarrays

Transfer RNAs (tRNA) are abundant short non-coding RNA species that are typically 76 to 90 nucleotides in length. tRNAs are directly responsible for protein synthesis by translating codons in mRNA into amino acid sequences. tRNAs were long considered as house-keeping molecules that lacked regulatory functions. However, a growing body of evidence indicates that cellular tRNA levels fluctuate in correspondence to varying conditions such as cell type, environment, and stress. The fluctuation of tRNA expression directly influences gene translation, favoring or repressing the expression of particular proteins. Ultimately comprehending the dynamic of protein synthesis requires the development of methods able to deliver high-quality tRNA profiles. The method that we present here is named SPOt, which stands for Streamlined Platform for Observing tRNA. SPOt consists of three steps starting with metabolic labeling of cell cultures with radioactive orthophosphate, followed by guanidinium thiocyanate-phenol-chloroform extraction of radioactive total RNAs and finally hybridization on in-house printed macroarrays. tRNA levels are estimated by quantifying the radioactivity intensities at each probe spot. In the protocol presented here we profile tRNAs in Mycobacterium smegmatis mc2155, a nonpathogenic bacterium often used as a model organism to study tuberculosis.

We describe an original transfer RNA analysis platform named SPOt (Streamlined Platform for Observing tRNA). SPOt simultaneously measures cellular levels of all tRNAs in biological samples, in only three steps, and in less than 24 hours.

使用 Macroarrays 的转移 rna 的代谢标记和剖面分析

Transfer RNAs (tRNA) are abundant short non-coding RNA species that are typically 76 to 90 nucleotides in length. tRNAs are directly responsible for ...