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Comprehensive qPCR profiling of gene expression in single neuronal cells

Nature Protocols volume 7, pages 118–127 (2012)Cite this article

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Abstract

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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Figure 1: A schematic workflow for single-cell gene profiling.
Figure 2: Micromanipulator-assisted manual collection of single neurons.
Figure 3: Assessment of primer efficiency and specificity.
Figure 4: Single-cell gene expression profiling using Fluidigm dynamic arrays.

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Acknowledgements

A.C. acknowledges the generous support of the AXA Research Fund. Z.P.P. is supported by the Brain and Behavior Research Foundation (National Alliance for Research on Schizophrenia and Depression (NARSAD) Young Investigator Award) and the Robert Wood Johnson Foundation. We thank N. Yang for help in experimental work. We thank S. Chavez for initial instruction in the use of the Fluidigm Biomark system, and R. Reijo Pera for access to the Fluidigm Biomark system in her lab. We also thank members of the Malenka and Südhof labs for their comments on the manuscript.

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  1. Ami Citri and Zhiping P Pang: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Psychiatry and Behavioral Sciences, Stanford University School of Medicine, Stanford, California, USA

    Ami Citri & Robert C Malenka

  2. Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, California, USA

    Zhiping P Pang & Thomas C Südhof

  3. Department of Neuroscience and Cell Biology, Child Health Institute of New Jersey, Robert Wood Johnson Medical School, New Brunswick, New Jersey, USA

    Zhiping P Pang

  4. Howard Hughes Medical Institute, Chevy Chase, Maryland, USA

    Thomas C Südhof

  5. Department of Pathology, Institute for Stem Cell Biology and Regenerative Medicine,

    Marius Wernig

  6. Stanford University School of Medicine, Stanford, California, USA

    Marius Wernig

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  1. Ami Citri
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Contributions

A.C. and Z.P.P. conceived the protocol, collected the data, presented the figures and wrote the manuscript. T.C.S., M.W. and R.C.M. supervised the project and contributed to the writing.

Corresponding authors

Correspondence to Ami Citri, Zhiping P Pang or Robert C Malenka.

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The authors declare no competing financial interests.

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Citri, A., Pang, Z., Südhof, T. et al. Comprehensive qPCR profiling of gene expression in single neuronal cells. Nat Protoc 7, 118–127 (2012). https://doi.org/10.1038/nprot.2011.430

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