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High-Throughput Single-Cell Real-Time Quantitative PCR Analysis

  • Liora Haim-Vilmovsky
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1979)

Abstract

Examining transcriptomics of populations at the single-cell level allows for higher resolution when studying functionality in development, differentiation, and physiology. Real-time quantitative PCR (qPCR) enables a sensitive detection of specific gene expression; however, processing a large number of samples for single-cell research involves a time-consuming process and high reagent costs. Here we describe a protocol for single-cell qPCR using nanofluidic chips. This method reduces the number of handling steps and volumes per reaction, allowing for more samples and genes to be measured.

Key words

Quantitative real-time PCR Single cells Targeted assays Gene expression Transcripts 

Notes

Acknowledgments

This work was supported by EMBO (award number ALTF 698-2012), Directorate-General for Research and Innovation (FP7-PEOPLE-2010-IEF, ThPLAST 274192) and an EMBL Interdisciplinary Postdoctoral fellowship, supported by H2020 Marie Skłodowska-Curie Actions.

References

  1. 1.
    Shen-Orr SS, Tibshirani R, Khatri P et al (2010) Cell type-specific gene expression differences in complex tissues. Nat Methods 7:287–289CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Hebenstreit D, Fang M, Gu M et al (2011) RNA sequencing reveals two major classes of gene expression levels in metazoan cells. Mol Syst Biol 7:497CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Hebenstreit D, Teichmann SA (2011) Analysis and simulation of gene expression profiles in pure and mixed cell populations. Phys Biol 8:035013CrossRefPubMedGoogle Scholar
  4. 4.
    Spurgeon SL, Jones RC, Ramakrishnan R (2008) High throughput gene expression measurement with real time PCR in a microfluidic dynamic array. PLoS One 3:e1662CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Jang JS, Simon VA, Feddersen RM et al (2011) Quantitative miRNA expression analysis using Fluidigm microfluidics dynamic arrays. BMC Genomics 12:144CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Devonshire AS, Elaswarapu R, Foy CA (2011) Applicability of RNA standards for evaluating RT-qPCR assays and platforms. BMC Genomics 12:118CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Bengtsson M, Hemberg M, Rorsman P, Ståhlberg A (2008) Quantification of mRNA in single cells and modelling of RT-qPCR induced noise. BMC Mol Biol 9:63CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Liora Haim-Vilmovsky
    • 1
    • 2
  1. 1.EMBL-European Bioinformatics InstituteWellcome Trust Genome CampusCambridgeUK
  2. 2.Wellcome Sanger InstituteWellcome Genome CampusCambridgeUK

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