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.
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References
Shen-Orr SS, Tibshirani R, Khatri P et al (2010) Cell type-specific gene expression differences in complex tissues. Nat Methods 7:287–289
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 7:497
Hebenstreit D, Teichmann SA (2011) Analysis and simulation of gene expression profiles in pure and mixed cell populations. Phys Biol 8:035013
Spurgeon SL, Jones RC, Ramakrishnan R (2008) High throughput gene expression measurement with real time PCR in a microfluidic dynamic array. PLoS One 3:e1662
Jang JS, Simon VA, Feddersen RM et al (2011) Quantitative miRNA expression analysis using Fluidigm microfluidics dynamic arrays. BMC Genomics 12:144
Devonshire AS, Elaswarapu R, Foy CA (2011) Applicability of RNA standards for evaluating RT-qPCR assays and platforms. BMC Genomics 12:118
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 9:63
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.
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Haim-Vilmovsky, L. (2019). High-Throughput Single-Cell Real-Time Quantitative PCR Analysis. In: Proserpio, V. (eds) Single Cell Methods. Methods in Molecular Biology, vol 1979. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9240-9_11
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DOI: https://doi.org/10.1007/978-1-4939-9240-9_11
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Publisher Name: Humana, New York, NY
Print ISBN: 978-1-4939-9239-3
Online ISBN: 978-1-4939-9240-9
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