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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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+quantitative+PCR+using+nested+primers.">Improved quantitative PCR using nested primers.</a> PCR Methods Appl. 3 (6), 332-337 (1994).</li><li>Hashimshony, T., Senderovich, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CEL-Seq2:+sensitive+highly-multiplexed+single-cell+RNA-Seq.">CEL-Seq2: sensitive highly-multiplexed single-cell RNA-Seq.</a> Genome Biol. 17, 77 (2016).</li><li>Ronander, E., Bengtsson, D. C., Joergensen, L., Jensen, A. T. R., Arnot, D. 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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?...

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

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

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 ...

科研

遗传学

Isolation and Characterization of Single Cells from Zebrafish Embryos

The zebrafish (Danio rerio) is a powerful model organism to study vertebrate development. Though many aspects of zebrafish embryonic development have been described at the morphological level, little is known about the molecular basis of cellular changes that occur as the organism develops. With recent advancements in microfluidics and multiplexing technologies, it is now possible to characterize gene expression in single cells. This allows for investigation of heterogeneity between individual cells of specific cell populations to identify and classify cell subtypes, characterize intermediate states that occur during cell differentiation, and explore differential cellular responses to stimuli. This study describes a protocol to isolate viable, single cells from zebrafish embryos for high throughput multiplexing assays. This method may be rapidly applied to any zebrafish embryonic cell type with fluorescent markers. An extension of this method may also be used in combination with high throughput sequencing technologies to fully characterize the transcriptome of single cells. As proof of principle, the relative abundance of cardiac differentiation markers was assessed in isolated, single cells derived from nkx2.5 positive cardiac progenitors. By evaluation of gene expression at the single cell level and at a single time point, the data support a model in which cardiac progenitors coexist with differentiating progeny. The method and work flow described here is broadly applicable to the zebrafish research community, requiring only a labeled transgenic fish line and access to microfluidics technologies.

This protocol describes a method for isolating single cells from zebrafish embryos, enriching for cells of interest, capturing zebrafish cells in microfluidic based single cell multiplex systems, and assessing gene expression from single cells.

分离和斑马鱼胚胎单细胞的表征

The zebrafish (Danio rerio) is a powerful model organism to study vertebrate development. Though many aspects of zebrafish embryonic development have been ...

发育生物学

A Combinatorial Single-cell Approach to Characterize the Molecular and Immunophenotypic Heterogeneity of Human Stem and Progenitor Populations

Immunophenotypic characterization and molecular analysis have long been used to delineate heterogeneity and define distinct cell populations. FACS is inherently a single-cell assay, however prior to molecular analysis, the target cells are often prospectively isolated in bulk, thereby losing single-cell resolution. Single-cell gene expression analysis provides a means to understand molecular differences between individual cells in heterogeneous cell populations. In bulk cell analysis an overrepresentation of a distinct cell type results in biases and occlusions of signals from rare cells with biological importance. By utilizing FACS index sorting coupled to single-cell gene expression analysis, populations can be investigated without the loss of single-cell resolution while cells with intermediate cell surface marker expression are also captured, enabling evaluation of the relevance of continuous surface marker expression. Here, we describe an approach that combines single-cell reverse transcription quantitative PCR (RT-qPCR) and FACS index sorting to simultaneously characterize the molecular and immunophenotypic heterogeneity within cell populations.
In contrast to single-cell RNA sequencing methods, the use of qPCR with specific target amplification allows for robust measurements of low-abundance transcripts with fewer dropouts, while it is not confounded by issues related to cell-to-cell variations in read depth. Moreover, by directly index-sorting single-cells into lysis buffer this method, allows for cDNA synthesis and specific target pre-amplification to be performed in one step as well as for correlation of subsequently derived molecular signatures with cell surface marker expression. The described approach has been developed to investigate hematopoietic single-cells, but have also been used successfully on other cell types.
In conclusion, the approach described herein allows for sensitive measurement of mRNA expression for a panel of pre-selected genes with the possibility to develop protocols for subsequent prospective isolation of molecularly distinct subpopulations.

Bulk gene expression measurements cloud individual cell differences in heterogeneous cell populations. Here, we describe a protocol for how single-cell gene expression analysis and index sorting by Florescence Activated Cell Sorting (FACS) can be combined to delineate heterogeneity and immunophenotypically characterize molecularly distinct cell populations.

一种组合单细胞法表征人茎和祖种群的分子和免疫表型异质性

Immunophenotypic characterization and molecular analysis have long been used to delineate heterogeneity and define distinct cell populations. FACS is ...

Single-cell Profiling of Developing and Mature Retinal Neurons

Highly specialized, but exceedingly small populations of cells play important roles in many tissues. The identification of cell-type specific markers and gene expression programs for extremely rare cell subsets has been a challenge using standard whole-tissue approaches. Gene expression profiling of individual cells allows for unprecedented access to cell types that comprise only a small percentage of the total tissue1-7. In addition, this technique can be used to examine the gene expression programs that are transiently expressed in small numbers of cells during dynamic developmental transitions8.
This issue of cellular diversity arises repeatedly in the central nervous system (CNS) where neuronal connections can occur between quite diverse cells9. The exact number of distinct cell types is not precisely known, but it has been estimated that there may be as many as 1000 different types in the cortex itself10. The function(s) of complex neural circuits may rely on some of the rare neuronal types and the genes they express. By identifying new markers and helping to molecularly classify different neurons, the single-cell approach is particularly useful in the analysis of cell types in the nervous system. It may also help to elucidate mechanisms of neural development by identifying differentially expressed genes and gene pathways during early stages of neuronal progenitor development.
As a simple, easily accessed tissue with considerable neuronal diversity, the vertebrate retina is an excellent model system for studying the processes of cellular development, neuronal differentiation and neuronal diversification. However, as in other parts of the CNS, this cellular diversity can present a problem for determining the genetic pathways that drive retinal progenitors to adopt a specific cell fate, especially given that rod photoreceptors make up the majority of the total retinal cell population11. Here we report a method for the identification of the transcripts expressed in single retinal cells (Figure 1). The single-cell profiling technique allows for the assessment of the amount of heterogeneity present within different cellular populations of the retina2,4,5,12. In addition, this method has revealed a host of new candidate genes that may play role(s) in the cell fate decision-making processes that occur in subsets of retinal progenitor cells8. With some simple adjustments to the protocol, this technique can be utilized for many different tissues and cell types.

A method for the isolation of single retinal cells and subsequent amplification of their cDNAs is described. Single-cell transcriptomics reveals the degree of cellular heterogeneity present in a tissue and uncovers new marker genes for rare cell populations. The accompanying protocol can be adjusted to suit many different cell types.

视网膜神经细胞发育和成熟的单细胞的剖析

Highly specialized, but exceedingly small populations of cells play important roles in many tissues. The identification of cell-type specific markers and ...

神经科学

Single Cell Analysis Of Transcriptionally Active Alleles By Single Molecule FISH

Gene transcription is an essential process in cell biology, and allows cells to interpret and respond to internal and external cues. Traditional bulk population methods (Northern blot, PCR, and RNAseq) that measure mRNA levels lack the ability to provide information on cell-to-cell variation in responses. Precise single cell and allelic visualization and quantification is possible via single molecule RNA fluorescence in situ hybridization (smFISH). RNA-FISH is performed by hybridizing target RNAs with labeled oligonucleotide probes. These can be imaged in medium/high throughput modalities, and, through image analysis pipelines, provide quantitative data on both mature and nascent RNAs, all at the single cell level. The fixation, permeabilization, hybridization and imaging steps have been optimized in the lab over many years using the model system described herein, which results in successful and robust single cell analysis of smFISH labeling. The main goal with sample preparation and processing is to produce high quality images characterized by a high signal-to-noise ratio to reduce false positives and provide data that are more accurate. Here, we present a protocol describing the pipeline from sample preparation to data analysis in conjunction with suggestions and optimization steps to tailor to specific samples.&#160;

Single molecule RNA fluorescence in situ hybridization (smFISH) is a method to accurately quantify levels and localization of specific RNAs at the single cell level. Here, we report our validated lab protocols for wet-bench processing, imaging and image analysis for single cell quantification of specific RNAs.

单分子鱼的转录活性等位基因单细胞分析

Gene transcription is an essential process in cell biology, and allows cells to interpret and respond to internal and external cues. Traditional bulk ...

生物化学

Gene Expression Profiling of Infecting Microbes Using a Digital Bar-coding Platform

For most mammalian pathogens, gene expression profiling studies have been limited by technical difficulties to accurately quantify pathogen gene transcripts from infected tissues. Pathogen RNA constitutes a tiny portion of the total RNA isolated from infected tissue samples. Both microarray and RNAseq technologies have difficulties in generating reliable reads for weakly expressed pathogen genes. Mutant pathogen strains with reduced in vivo proliferation pose an even bigger challenge. Here we describe an in vivo gene expression profiling protocol that is very fast, extremely sensitive and highly reproducible. We developed this protocol during our investigation of the fungal pathogen Candida albicans in a murine model of hematogenously disseminated candidiasis. Using this protocol, we have documented time courses of dynamically regulated C. albicans gene expression during kidney infection, and discovered unexpected features of gene expression responses to antifungal drug treatment in vivo.

We developed a protocol that is fast, sensitive and reproducible for pathogen gene expression profiling during an infection.

侵染微生物利用数字条码平台的基因表达谱

For most mammalian pathogens, gene expression profiling studies have been limited by technical difficulties to accurately quantify pathogen gene ...

免疫与感染

Fluorescence Activated Cell Sorting (FACS) and Gene Expression Analysis of Fos-expressing Neurons from Fresh and Frozen Rat Brain Tissue

The study of neuroplasticity and molecular alterations in learned behaviors is switching from the study of whole brain regions to the study of specific sets of sparsely distributed activated neurons called neuronal ensembles that mediate learned associations. Fluorescence Activated Cell Sorting (FACS) has recently been optimized for adult rat brain tissue and allowed isolation of activated neurons using antibodies against the neuronal marker NeuN and Fos protein, a marker of strongly activated neurons. Until now, Fos-expressing neurons and other cell types were isolated from fresh tissue, which entailed long processing days and allowed very limited numbers of brain samples to be assessed after lengthy and complex behavioral procedures. Here we found that yields of Fos-expressing neurons and Fos mRNA from dorsal striatum were similar between freshly dissected tissue and tissue frozen at -80 &#186;C for 3 - 21 days. In addition, we confirmed the phenotype of the NeuN-positive and NeuN-negative sorted cells by assessing gene expression of neuronal (NeuN), astrocytic (GFAP), oligodendrocytic (Oligo2) and microgial (Iba1) markers, which indicates that frozen tissue can also be used for FACS isolation of glial cell types. Overall, it is possible to collect, dissect and freeze brain tissue for multiple FACS sessions. This maximizes the amount of data obtained from valuable animal subjects that have often undergone long and complex behavioral procedures.

Fluorescence Activated Cell Sorting FACS and Gene Expression Analysis of Fos-expressing Neurons from Fresh and Frozen Rat Brain Tissue

Here we present a Fluorescence Activated Cell Sorting (FACS) protocol to study molecular alterations in Fos-expressing neuronal ensembles from both fresh and frozen brain tissue. The use of frozen tissue allows FACS isolation of many brain areas over multiple sessions to maximize the use of valuable animal subjects.

荧光活化细胞分选（FACS）和新鲜和冷冻大鼠脑组织的Fos表达神经元基因表达分析

The study of neuroplasticity and molecular alterations in learned behaviors is switching from the study of whole brain regions to the study of specific ...

Single Cell Transcriptional Profiling of Adult Mouse Cardiomyocytes

While numerous studies have examined gene expression changes from homogenates of heart tissue, this prevents studying the inherent stochastic variation between cells within a tissue. Isolation of pure cardiomyocyte populations through a collagenase perfusion of mouse hearts facilitates the generation of single cell microarrays for whole transcriptome gene expression, or qPCR of specific targets using nanofluidic arrays. We describe here a procedure to examine single cell gene expression profiles of cardiomyocytes isolated from the heart. This paradigm allows for the evaluation of metrics of interest which are not reliant on the mean (for example variance between cells within a tissue) which is not possible when using conventional whole tissue workflows for the evaluation of gene expression (Figure 1). We have achieved robust amplification of the single cell transcriptome yielding micrograms of double stranded cDNA that facilitates the use of microarrays on individual cells. In the procedure we describe the use of NimbleGen arrays which were selected for their ease of use and ability to customize their design. Alternatively, a reverse transcriptase - specific target amplification (RT-STA) reaction, allows for qPCR of hundreds of targets by nanofluidic PCR. Using either of these approaches, it is possible to examine the variability of expression between cells, as well as examining expression profiles of rare cell types from within a tissue. Overall, the single cell gene expression approach allows for the generation of data that can potentially identify idiosyncratic expression profiles that are typically averaged out when examining expression of millions of cells from typical homogenates generated from whole tissues.

Single cell expression profiling allows the detailed gene expression analysis of individual cells. We describe methods for the isolation of cardiomyocytes, and preparing the resulting lysates for either whole transcriptome microarray or qPCR of specific targets.

成年小鼠心肌细胞的单细胞转录谱

While numerous studies have examined gene expression changes from homogenates of heart tissue, this prevents studying the inherent stochastic variation ...

生物学

Analysis of Single-cell Gene Transcription by RNA Fluorescent In Situ Hybridization (FISH)

Adhesion of Plasmodium falciparum infected erythrocytes (IE) to human endothelial receptors during malaria infections is mediated by expression of PfEMP1 protein variants encoded by the var genes.
The haploid P. falciparum genome harbors approximately 60 different var genes of which only one has been believed to be transcribed per cell at a time during the blood stage of the infection. How such mutually exclusive regulation of var gene transcription is achieved is unclear, as is the identification of individual var genes or sub-groups of var genes associated with different receptors and the consequence of differential binding on the clinical outcome of P. falciparum infections. Recently, the mutually exclusive transcription paradigm has been called into doubt by transcription assays based on individual P. falciparum transcript identification in single infected erythrocytic cells using RNA fluorescent in situ hybridization (FISH) analysis of var gene transcription by the parasite in individual nuclei of P. falciparum IE1.
Here, we present a detailed protocol for carrying out the RNA-FISH methodology for analysis of var gene transcription in single-nuclei of P. falciparum infected human erythrocytes. The method is based on the use of digoxigenin- and biotin- labeled antisense RNA probes using the TSA Plus Fluorescence Palette System2 (Perkin Elmer), microscopic analyses and freshly selected P. falciparum IE. The in situ hybridization method can be used to monitor transcription and regulation of a variety of genes expressed during the different stages of the P. falciparum life cycle and is adaptable to other malaria parasite species and other organisms and cell types.

Analysis of Single-cell Gene Transcription by RNA Fluorescent In Situ Hybridization FISH

Fluorescent in situ hybridization (FISH) to identify mRNA transcripts in individual cells allows analysis of polygenic activity such as the simultaneous transcription of more than one member of the var multigene family in Plasmodium falciparum infected erythrocytes 1. The technique is adaptable and can be used on different types of genes, cells and organisms.

单细胞基因转录的RNA荧光分析<em在原位</em&gt;杂交（FISH）

Adhesion of Plasmodium falciparum infected erythrocytes (IE) to human endothelial receptors during malaria infections is mediated by expression of PfEMP1 ...

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for using FISH to quantify the number of mRNAs in single yeast cells. Cells can be grown in any condition of interest and then fixed and made permeable. Subsequently, multiple single-stranded deoxyoligonucleotides conjugated to fluorescent dyes are used to label and visualize mRNAs. Diffraction-limited fluorescence from single mRNA molecules is quantified using a spot-detection algorithm to identify and count the number of mRNAs per cell. While the more standard quantification methods of northern blots, RT-PCR and gene expression microarrays provide information on average mRNAs in the bulk population, FISH facilitates both the counting and localization of these mRNAs in single cells at single-molecule resolution.

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

This protocol describes an experimental procedure for performing Fluorescence in situ Hybridization (FISH) for counting mRNAs in single cells at single-molecule resolution.

可视化和分析的mRNA分子，使用荧光<em&gt;在原位</em&gt;杂交<em&gt;酿酒酵母（Saccharomyces cerevisiae）</em

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for ...

单细胞基因表达谱使用流式细胞仪和qPCR内部标准

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