Comprehensive qPCR profiling of gene expression in single neuronal cells

Department of Psychiatry and Behavioral Sciences, Stanford University School of Medicine, California, USA.
Nature Protocol (Impact Factor: 9.67). 01/2012; 7(1):118-27. DOI: 10.1038/nprot.2011.430
Source: PubMed


A major challenge in neuronal stem cell biology lies in characterization of lineage-specific reprogrammed human neuronal cells, a process that necessitates the use of an assay sensitive to the single-cell level. Single-cell gene profiling can provide definitive evidence regarding the conversion of one cell type into another at a high level of resolution. The protocol we describe uses Fluidigm Biomark dynamic arrays for high-throughput expression profiling from single neuronal cells, assaying up to 96 independent samples with up to 96 quantitative PCR (qPCR) probes (equivalent to 9,216 reactions) in a single experiment, which can be completed within 2-3 d. The protocol enables simple and cost-effective profiling of several hundred transcripts from a single cell, and it could have numerous utilities.

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    • "0.5 µg of total RNA was processed with the First Strand cDNA Synthesis Kit according to the manufacturer’s protocol (Roche Applied Science) and RT-qPCR was performed for the 19 genes showing at least 2-fold expression differences in the microarray analysis (Taqman gene expression assays used are listed in Table S2) using the Biomark 96.96 Dynamic Arrays Fluidigm RT-qPCR platform [14]. Relative gene expressions were calculated with the 2−ΔΔCt method [15], using household gene Beta Actin (ACTB) expression as internal standard. "
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    ABSTRACT: Objective Identify gene expression profiles associated with OA processes in articular cartilage and determine pathways changing during the disease process. Methods Genome wide gene expression was determined in paired samples of OA affected and preserved cartilage of the same joint using microarray analysis for 33 patients of the RAAK study. Results were replicated in independent samples by RT-qPCR and immunohistochemistry. Profiles were analyzed with the online analysis tools DAVID and STRING to identify enrichment for specific pathways and protein-protein interactions. Results Among the 1717 genes that were significantly differently expressed between OA affected and preserved cartilage we found significant enrichment for genes involved in skeletal development (e.g. TNFRSF11B and FRZB). Also several inflammatory genes such as CD55, PTGES and TNFAIP6, previously identified in within-joint analyses as well as in analyses comparing preserved cartilage from OA affected joints versus healthy cartilage were among the top genes. Of note was the high up-regulation of NGF in OA cartilage. RT-qPCR confirmed differential expression for 18 out of 19 genes with expression changes of 2-fold or higher, and immunohistochemistry of selected genes showed a concordant change in protein expression. Most of these changes associated with OA severity (Mankin score) but were independent of joint-site or sex. Conclusion We provide further insights into the ongoing OA pathophysiological processes in cartilage, in particular into differences in macroscopically intact cartilage compared to OA affected cartilage, which seem relatively consistent and independent of sex or joint. We advocate that development of treatment could benefit by focusing on these similarities in gene expression changes and/or pathways.
    PLoS ONE 07/2014; 9(7):e103056. DOI:10.1371/journal.pone.0103056 · 3.23 Impact Factor
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    • "To determine molecular level identification and gene signatures that encompass antigen specificity, a Fluidigm Biomark dynamic array could be introduced [82]. This qPCR-base single-cell gene-profiling technique can assay up to 96 genes from 96 individual cells in a single experiment. "
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    ABSTRACT: More than 60 million people in the world have been diagnosed with human immunodeficiency virus (HIV) infections since the virus was recognized as the causative agent of acquired immunodeficiency syndrome (AIDS) in the 1980s. Even though more than half of the infected patients have died, effective disease treatment and prevention measures have not been established. Antiretroviral therapy is the only proven HIV treatment which sustains suppression of patient viremia. Current routine approaches to treat HIV infections are targeted at developing vaccines that will induce humoral or cell memory immune responses. However, developing an effective vaccine has been challenging because the HIV mutates rapidly, which allows the virus to evade immune surveillances established against the previous strain. In addition, the virus is able to quickly establish a reservoir and treatment is difficult because of the general lack of knowledge about HIV immune response mechanisms. This review introduces common disease symptoms and the progression of HIV infection with a brief summary of the current treatment approaches. Different cellular immune responses against HIV are also discussed, with emphasis on a nanotechnology research that has focused on probing T cell response to HIV infection. Furthermore, we discuss recent noteworthy nanotechnology updates on T cell response screening that are focused on HIV infection. Finally, we review potential future treatment strategies based on the correlations between T cell response and HIV infection.
    Bioscience Reports 07/2014; 34(4). DOI:10.1042/BSR20140097 · 2.64 Impact Factor
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    • "One may wonder whether, in reality, we are generating a mixed population of OLs, and whether multiple subtypes of OPCs and OLs exist and can be recognized in vitro. Following the example of neuronal studies, single cell gene expression profiling could help addressing this question (Citri et al., 2012). "
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    ABSTRACT: Since monumental studies from scientists like His, Ramón y Cajal, Lorente de Nó and many others have put down roots for modern neuroscience, the scientific community has spent a considerable amount of time, and money, investigating any aspect of the evolution, development and function of neurons. Today, the complexity and diversity of myriads of neuronal populations is still focus of extensive studies in hundreds of laboratories around the world. However, our prevalent neuron-centric perspective has dampened the efforts in understanding glial cells, even though their active participation in the brain physiology and pathophysiology has been increasingly recognized over the years. Among all glial cells of the central nervous system (CNS), oligodendrocytes (OLs) are a particularly specialized type of cells that provide fundamental support to neuronal activity by producing the myelin sheath. Despite their functional relevance, the developmental mechanisms regulating the generation of OLs are still poorly understood. In particular, it is still not known whether these cells share the same degree of heterogeneity of their neuronal companions and whether multiple subtypes exist within the lineage. Here, we will review and discuss current knowledge about OL development and function in the brain and spinal cord. We will try to address some specific questions: do multiple OL subtypes exist in the CNS? What is the evidence for their existence and those against them? What are the functional features that define an oligodendrocyte? We will end our journey by reviewing recent advances in human pluripotent stem cell differentiation towards OLs. This exciting field is still at its earliest days, but it is quickly evolving with improved protocols to generate functional OLs from different spatial origins. As stem cells constitute now an unprecedented source of human OLs, we believe that they will become an increasingly valuable tool for deciphering the complexity of human OL identity.
    Frontiers in Cellular Neuroscience 05/2014; 8. DOI:10.3389/fncel.2014.00201 · 4.29 Impact Factor
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