Single-Cell PCR Profiling of Gene Expression in Hematopoiesis

Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1185)

Abstract

Single-cell analysis of gene expression offers the possibility of exploring cellular and molecular heterogeneity in stem and developmental cell systems, including cancer, to infer routes of cellular specification and their respective gene regulatory modules. PCR-based technologies, although limited to the analysis of a predefined set of genes, afford a cost-effective balance of throughput and biological information and have become a method of choice in stem cell laboratories. Here we describe an experimental and analytical protocol based on the Fluidigm microfluidics platform for the simultaneous expression analysis of 48 or 96 genes in multiples of 48 or 96 cells. We detail wet laboratory procedures and describe clustering, principal component analysis, correlation, and classification tools for the inference of cellular pathways and gene networks.

Key words

Single-cell quantitative RT-PCR Microfluidics Hierarchical clustering Principal component analysis Machine learning Random forests Logistic regression Correlation-based gene networks 

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Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  1. 1.Stem Cell LaboratoryUniversity College London Cancer InstituteLondonUK
  2. 2.Computational Biology and Biological Physics, Department of Astronomy and Theoretical PhysicsLund UniversityLundSweden
  3. 3.Department of Haematology, NHS-Blood and TransplantUniversity of CambridgeCambridgeUK

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