Single-cell Microarray Research Articles

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 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.

Dpy19l1, a multi-transmembrane protein, regulates the radial migration of glutamatergic neurons in the developing cerebral cortex

During corticogenesis, the regulation of neuronal migration is crucial for the functional organization of the neocortex. Glutamatergic neurons are major excitatory components of the mammalian neocortex. In order to elucidate the specific molecular mechanisms underlying their development, we used single-cell microarray analysis to screen for mouse genes that are highly expressed in developing glutamatergic neurons. We identified dpy-19-like 1 (Dpy19l1), a homolog of C. elegans dpy-19, which encodes a putative multi-transmembrane protein shown to regulate directed migration of Q neuroblasts in C. elegans. At embryonic stages Dpy19l1 is highly expressed in glutamatergic neurons in the mouse cerebral cortex, whereas in the subpallium, where GABAergic neurons are generated, expression was below detectable levels. Downregulation of Dpy19l1 mediated by shRNA resulted in defective radial migration of glutamatergic neurons in vivo, which was restored by the expression of shRNA-insensitive Dpy19l1. Many Dpy19l1-knockdown cells were aberrantly arrested in the intermediate zone and the deep layer and, additionally, some extended single long processes towards the pial surface. Furthermore, we observed defective radial migration of bipolar cells in Dpy19l1-knockdown brains. Despite these migration defects, these cells correctly expressed Cux1, which is a marker for upper layer neurons, suggesting that Dpy19l1 knockdown results in migration defects but does not affect cell type specification. These results indicate that Dpy19l1 is required for the proper radial migration of glutamatergic neurons, and suggest an evolutionarily conserved role for the Dpy19 family in neuronal migration.

Transcript amplification from single bacterium for transcriptome analysis

Total transcript amplification (TTA) from single eukaryotic cells for transcriptome analysis is established, but TTA from a single prokaryotic cell presents additional challenges with much less starting material, the lack of poly(A)-tails, and the fact that the messages can be polycistronic. Here, we describe a novel method for single-bacterium TTA using a model organism, Burkholderia thailandensis, exposed to a subinhibitory concentration of the antibacterial agent, glyphosate. Utilizing a B. thailandensis microarray to assess the TTA method showed low fold-change bias (less than twofold difference and Pearson correlation coefficient R ≈ 0.87-0.89) and drop-outs (4%-6% of 2842 detectable genes), compared with data obtained from the larger-scale nonamplified RNA samples. Further analysis of the microarray data suggests that B. thailandensis, when exposed to the aromatic amino acid biosynthesis inhibitor glyphosate, induces (or represses) genes to possibly recuperate and balance the intracellular amino acid pool. We validated our single-cell microarray data at the multi-cell and single-cell levels with lacZ and gfp reporter-gene fusions, respectively. Sanger sequencing of 192 clones generated from the TTA product of a single cell, with and without enrichment by elimination of rRNA and tRNA, detected only B. thailandensis sequences with no contamination. These data indicate that RNA-seq of TTA from a single cell is possible using this novel method.

Preclinical validation of a microarray method for full molecular karyotyping of blastomeres in a 24-h protocol

BACKGROUNDPreimplantation genetic screening (PGS) has been used in an attempt to determine embryonic aneuploidy. Techniques that use new molecular methods to determine the karyotype of an embryo are expanding the scope of PGS.METHODSWe introduce a new method for PGS, termed ‘parental support’, which leverages microarray measurements from parental DNA to ‘clean’ single-cell microarray measurements on embryonic cells and explicitly computes confidence in each copy number call. The method distinguishes mitotic and meiotic copy errors and determines parental source of aneuploidy.RESULTSValidation with 459 single cells of known karyotype indicated that per-cell false-positive and false-negative rates are roughly equivalent to the ‘gold standard’ metaphase karyotype. The majority of the cells were run in parallel with a clinical commercial PGS service. Computed confidences were conservative and roughly concordant with accuracy. To examine ploidy in human embryos, the method was then applied to 26 disaggregated, cryopreserved, cleavage-stage embryos for a total of 134 single blastomeres. Only 23.1% of the embryos were euploid, though 46.2% of embryos were mosaic euploid. Mosaicism affected 57.7% of the embryos. Counts of mitotic and meiotic errors were roughly equivalent. Maternal meiotic trisomy predominated over paternal trisomy, and maternal meiotic trisomies were negatively predictive of mosaic euploid embryos.CONCLUSIONSWe have performed a major preclinical validation of a new method for PGS and found that the technology performs approximately as well as a metaphase karyotype. We also directly measured the mechanism of aneuploidy in cleavage-stage human embryos and found high rates and distinct patterns of mitotic and meiotic aneuploidy.

Development of Cell Chip System for Cytological Analysis and Diagnosis

Microchip technologies are expected to perform high-throughput analysis of the function of single-cells. In previous our study, we already developed a single-cell microarray chip for high-throughput screening and analysis of antigen-specific single B-cells. Using the technology of cell microarray chip, we have developed a new system for the high-sensitive and rapid diagnosis of parasitosis. The microarray chip is made from polystyrene with over 10,000 microchambers, which can accommodate 100 erythrocytes. As the result, over 1,000,000 erythrocytes were dispersed on a microarray. We could detect parasites as low as 0.0001% with spreading of human erythrocytes on the microarray followed within 15 min.

Conditional knockdown of Nanog induces apoptotic cell death in mouse migrating primordial germ cells

The pluripotency factor Nanog is expressed in peri-implantation embryos and primordial germ cells (PGCs). Nanog-deficient mouse embryos die soon after implantation. To explore the function of Nanog in germ cells, Nanog RNA was conditionally knocked down in vivo by shRNA. Nanog shRNA transgenic (NRi-Tg) mice were generated through the formation of germline chimeras with NRi-Tg embryonic stem cells. In E12.5 Cre-induced ER-Cre/NRi-Tg and TNAP-Cre/NRi-Tg double-transgenic embryos, the number of alkaline phosphatase-positive and SSEA1-positive PGCs decreased significantly. In the E9.5 and E10.5 migrating Nanog-knockdown PGCs, TUNEL-positive apoptotic cell death became prominent in vivo and in vitro, despite Oct4 expression. Single-cell microarray analysis of E10.5 Nanog-knockdown PGCs revealed significant up- and downregulation of a substantial number of genes, including Tial1, Id1 and Suz12. These data suggest that Nanog plays a key role in the proliferation and survival of migrating PGCs as a safeguard of the PGC-specific molecular network.

RoboSCell: an automated single cell arraying and analysis instrument

Single cell research has the potential to revolutionize experimental methods in biomedical sciences and contribute to clinical practices. Recent studies suggest analysis of single cells reveals novel features of intracellular processes, cell-to-cell interactions and cell structure. The methods of single cell analysis require mechanical resolution and accuracy that is not possible using conventional techniques. Robotic instruments and novel microdevices can achieve higher throughput and repeatability; however, the development of such instrumentation is a formidable task. A void exists in the state-of-the-art for automated analysis of single cells. With the increase in interest in single cell analyses in stem cell and cancer research the ability to facilitate higher throughput and repeatable procedures is necessary. In this paper, a high-throughput, single cell microarray-based robotic instrument, called the RoboSCell, is described. The proposed instrument employs a partially transparent single cell microarray (SCM) integrated with a robotic biomanipulator for in vitro analyses of live single cells trapped at the array sites. Cells, labeled with immunomagnetic particles, are captured at the array sites by channeling magnetic fields through encapsulated permalloy channels in the SCM. The RoboSCell is capable of systematically scanning the captured cells temporarily immobilized at the array sites and using optical methods to repeatedly measure extracellular and intracellular characteristics over time. The instrument's capabilities are demonstrated by arraying human T lymphocytes and measuring the uptake dynamics of calcein acetoxymethylester--all in a fully automated fashion.

Transcriptome transfer produces a predictable cellular phenotype

Cellular phenotype is the conglomerate of multiple cellular processes involving gene and protein expression that result in the elaboration of a cell's particular morphology and function. It has been thought that differentiated postmitotic cells have their genomes hard wired, with little ability for phenotypic plasticity. Here we show that transfer of the transcriptome from differentiated rat astrocytes into a nondividing differentiated rat neuron resulted in the conversion of the neuron into a functional astrocyte-like cell in a time-dependent manner. This single-cell study permits high resolution of molecular and functional components that underlie phenotype identity. The RNA population from astrocytes contains RNAs in the appropriate relative abundances that give rise to regulatory RNAs and translated proteins that enable astrocyte identity. When transferred into the postmitotic neuron, the astrocyte RNA population converts 44% of the neuronal host cells into the destination astrocyte-like phenotype. In support of this observation, quantitative measures of cellular morphology, single-cell PCR, single-cell microarray, and single-cell functional analyses have been performed. The host-cell phenotypic changes develop over many weeks and are persistent. We call this process of RNA-induced phenotype changes, transcriptome-induced phenotype remodeling.

Densely-Packed Microbowl Array with Balanced Dielectrophoretic Forces for Single-Cell Microarray

AbstractIn this paper, we demonstrate a perfectly-ordered microbowl array with balanced dielectrophoresis (DEP) for a high-throughput single-cell analysis. In order to fabricate well-ordered microbowl array in a large area, we utilized three-dimensional diffuser lithography for photoresist mold and nickel electroplating technique for final microbowl structures on a silicon substrate. Single microbowl has six sharp apexes surrounding the microbowl perimeter. Each microbowl has a diameter of 10 μm, and a height of 9 μm, which can be controllable by patterns on mask and lithography conditions. To investigate feasibility for application to the microbowl array as a single-cell microarray, we used latex beads of 6.4 μm in an average diameter to be captured by dielectrophoretic force. The nickel microbowl array densely packed with a hexagonal geometry played as a bottom electrode, and an ITO-coated glass covered the nickel microbowl array as a top electrode while keeping a uniform gap between two electrodes. After injecting solution containing latex beads through the gap, we applied an AC signal (2 VPP, 1 MHz) between two electrodes to induce high electric field near the sharp apexes of the single microbowl. A negative DEP trap is formed at the center of the single microbowl with balanced DEP force from the six apexes. The experimental result shows that injected latex beads had been successfully and uniformly aligned and trapped at the microbowl array sustained by negative DEP.

Complex genome-wide transcription dynamics orchestrated by Blimp1 for the specification of the germ cell lineage in mice

Specification of germ cell fate is fundamental in development. With a highly representative single-cell microarray and rigorous quantitative PCR analysis, we defined the genome-wide transcription dynamics that create primordial germ cells (PGCs) from the epiblast, a process that exclusively segregates them from their somatic neighbors. We also analyzed the effect of the loss of Blimp1, a key transcriptional regulator, on these dynamics. Our analysis revealed that PGC specification involves complex, yet highly ordered regulation of a large number of genes, proceeding under the strong influence of mesoderm induction but specifically avoiding developmental programs such as the epithelial-mesenchymal transition, Hox cluster activation, cell cycle progression, and DNA methyltransferase machinery. Remarkably, Blimp1 is essential for repressing nearly all the genes normally down-regulated in PGCs relative to their somatic neighbors. In contrast, it is dispensable for the activation of approximately half of the genes up-regulated in PGCs, uncovering the Blimp1-independent events for PGC specification. Notably, however, highly PGC-specific genes exhibited distinct correlations to Blimp1 in wild-type embryos, and these correlations faithfully predicted their expression impairments in Blimp1 mutants. Moreover, their expression overlaps within single cells were severely damaged without Blimp1, demonstrating that Blimp1 exerts positive influence on their concerted activation. Thus, Blimp1 is not a single initiator but a dominant coordinator of the transcriptional program for the establishment of the germ cell fate in mice.

Modelling and measuring single cell RNA expression levels find considerable transcriptional differences among phenotypically identical cells.

BackgroundPhenotypically identical cells demonstrate predictable, robust behaviours. However, there is uncertainty as to whether phenotypically identical cells are equally similar at the underlying transcriptional level or if cellular systems are inherently noisy. To answer this question, it is essential to distinguish between technical noise and true variation in transcript levels. A critical issue is the contribution of sampling effects, introduced by the requirement to globally amplify the single cell mRNA population, to observed measurements of relative transcript abundance.ResultsWe used single cell microarray data to develop simple mathematical models, ran Monte Carlo simulations of the impact of technical and sampling effects on single cell expression data, and compared these with experimental microarray data generated from single embryonic neural stem cells in vivo. We show that the actual distribution of measured gene expression ratios for pairs of neural stem cells is much broader than that predicted from our sampling effect model.ConclusionOur results confirm that significant differences in gene expression levels exist between phenotypically identical cells in vivo, and that these differences exceed any noise contribution from global mRNA amplification.

Method for single-cell microarray analysis and application to gene-expression profiling of GABAergic neuron progenitors

The mammalian central nervous system is populated with various types of neurons and glia. To investigate the functions and development of individual cells requires gene-expression analysis at the single-cell level. Here, we developed a microarray-based method for the gene-expression profiling of single cells and tested it for GABAergic neuron progenitors. Single GABAergic neuron progenitors were collected from the neocortex of GAD67-GFP knock-in mice by dissociation followed by the aspiration of GFP-positive cells. Complementary DNA from the single cells was amplified by a method in which Super SMART PCR and T7 RNA polymerase amplification were combined at a optimized condition. The cRNA was subjected to microarray hybridization and analysis, which yielded reliable and reproducible results.

Wnt4 Is a Local Repulsive Cue that Determines Synaptic Target Specificity

How synaptic specificity is molecularly coded in target cells is a long-standing question in neuroscience. Whereas essential roles of several target-derived attractive cues have been shown, less is known about the role of repulsion by nontarget cells. We conducted single-cell microarray analysis of two neighboring muscles (M12 and M13) in Drosophila, which are innervated by distinct motor neurons, by directly isolating them from dissected embryos. We identified a number of potential target cues that are differentially expressed between the two muscles, including M13-enriched Wnt4. When the functions of Wnt4, or putative receptors Frizzled 2 and Derailed-2 or Dishevelled were inhibited, motor neurons that normally innervate M12 (MN12s) formed smaller synapses on M12 but instead formed ectopic nerve endings on M13. Conversely, ectopic expression of Wnt4 in M12 inhibits synapse formation by MN12s. These results suggest that Wnt4, via Frizzled 2, Derailed-2, and Dishevelled, generates target specificity by preventing synapse formation on a nontarget muscle. Ectopic expression of five other M13-enriched genes, including beat-IIIc and Glutactin, also inhibits synapse formation by MN12s. These results demonstrate an important role for local repulsion in regulating cell-to-cell target specificity.

Time-resolved gene expression experiments by using a single-cell electroporation microarray

Time-resolved gene expression experiments by using a single-cell electroporation microarray

The mammalian transcriptome and the cellular complexity of the brain

The mammalian transcriptome and the cellular complexity of the brain

Comparative evaluation of linear and exponential amplification techniques for expression profiling at the single-cell level

Comparison of the performance of three methods for amplifying single-cell amounts of RNA for use in expression profiling shows that PCT amplification is more reliable than linear amplification.

Single-Cell Microarray for Analyzing Cellular Response

Detection of cellular response by measuring intracellular calcium, (Ca2+)i with Ca2+-dependent fluorescent dye are standard approaches to detect ligand-stimulated cells and to study signaling through ligand/receptor interaction. We describe a single-cell microarray system to analyze cellular response of individual cells such as lymphocytes using microchamber array chips. The single-cell microarray chip is made from polystyrene with over 30,000 microchambers, which can accommodate only single cells. Lymphocytes derived from mouse spleen or human blood were spread on the microarray, and over 80% of the microchambers achieved single-cell status. Stimulation of B-cells through antigen receptors on the microarray allowed us to detect activated B-cells by comparing the states of single B-cells before and after stimulation with antigen, which is disabled for flow cytometry. In addition, this novel method demonstrated retrieval of positive single B-cells from microchambers by a micromanipulator and achieved antibody DNA analysis. The system is suitable for high-throughput analysis of intracellular Ca2+ response at the single-cell level and is applicable to screen antigen-specific lymphocytes for making specific monoclonal antibody.

Single-cell transcriptional profiles and spatial patterning of the mammalian olfactory epithelium

In order to gain insights into the regulatory control of neuronal diversity in the mammalian olfactory system, we have identified the transcriptional profile of individual olfactory neurons. A single cell microarray strategy was performed to search for candidate genes involved in the molecular specification of dorso-ventral zones of olfactory receptor (OR) expression. Several transcripts were identified that display differential expression in distinct OR zones, including a novel family of genes, the Lozenge-like (Lzl) genes which share sequence consensus motifs with Lozenge, a transcription factor involved in the patterning of the Drosophila olfactory and visual systems.

Multilineage gene expression in human bone marrow stromal cells as evidenced by single-cell microarray analysis☆

The nonhematopoietic stromal cells of the bone marrow are critical for the development of hematopoietic stem cells into functionally competent blood cells. This study addresses the question of whether bone marrow stromal cell cultures in the Dexter system propagate multiple different mesenchymal stromal cell types or one stromal cell type that expresses multiple phenotypes. Results show that isolated single stromal cells simultaneously express transcripts associated with osteoblast, fibroblast, muscle, and adipocyte differentiation. Furthermore, isolated single stromal cells simultaneously express transcripts characteristic of epithelial cells, endothelial cells, and neural/glial cells. Isolated single stromal cells also express transcripts for CD45, CD19, CD10, CD79a, and representative proto-oncogenes and transcription factors, which are typically associated with normal and neoplastic hematopoietic cells. These findings suggest that the nonhematopoietic mesenchymal cells and the hematopoietic B-lymphocytes have a common progenitor. This is consistent with the idea that progenitor cells express genes that are characteristic of the multiple lineage paths that such cells may be capable of adopting. This study demonstrates the technical feasibility of transcriptome analysis of individual primary cell-culture grown stromal cells and supports the concept that bone marrow stromal cells are relatively homogeneous and show a phenotypic signature of potential multilineage differentiation capacity.