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Digital PCR pp 423–444Cite as

Very Low Abundance Single-Cell Transcript Quantification with 5-Plex ddPCRTM Assays

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 1768))

Abstract

Gene expression studies have provided one of the most accessible windows for understanding the molecular basis of cell and tissue phenotypes and how these change in response to stimuli. Current PCR-based and next generation sequencing methods offer great versatility in allowing the focused study of the roles of small numbers of genes or comprehensive profiling of the entire transcriptome of a sample at one time. Marrying of these approaches to various cell sorting technologies has recently enabled the profiling of expression in single cells, thereby increasing the resolution and sensitivity and strengthening the inferences from observed expression levels and changes. This chapter presents a quick and efficient 1-day workflow for sorting single cells with a small laboratory cell-sorter followed by an ultrahigh sensitivity, multiplexed digital PCR method for quantitative tracking of changes in 5–10 genes per single cell.

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Acknowledgments

We wish to thank our many colleagues who have assisted in the development of the instrumentation and reagents necessary to develop this protocol, especially Shenglong Wang who began earlier single-cell work from which this was developed. Special thanks also to Svilen Tzonev, Doug Hauge, Niels Klitgord, and Dimitri Skvortsov for assistance with the quantification analysis, and to Marcos Oquendo for assistance with the S3e cell sorter.

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Authors and Affiliations

  1. Digital Biology Center, Bio-Rad Laboratories, Pleasanton, CA, USA

    George Karlin-Neumann, Bin Zhang & Claudia Litterst

Authors
  1. George Karlin-Neumann
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  2. Bin Zhang
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  3. Claudia Litterst
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Corresponding author

Correspondence to George Karlin-Neumann .

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Editors and Affiliations

  1. Digital Biology Center, Bio-Rad Laboratories, Pleasanton, California, USA

    George Karlin-Neumann

  2. Digital Biology Center, Bio-Rad Laboratories, Pleasanton, California, USA

    Francisco Bizouarn

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Karlin-Neumann, G., Zhang, B., Litterst, C. (2018). Very Low Abundance Single-Cell Transcript Quantification with 5-Plex ddPCRTM Assays. In: Karlin-Neumann, G., Bizouarn, F. (eds) Digital PCR. Methods in Molecular Biology, vol 1768. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7778-9_24

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  • DOI: https://doi.org/10.1007/978-1-4939-7778-9_24

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7776-5

  • Online ISBN: 978-1-4939-7778-9

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