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Microfluidic single-cell real-time PCR for comparative analysis of gene expression patterns

Abstract

Single-cell quantitative real-time PCR (qRT-PCR) combined with high-throughput arrays allows the analysis of gene expression profiles at a molecular level in approximately 11 h after cell sample collection. We present here a high-content microfluidic real-time platform as a powerful tool for comparatively investigating the regulation of developmental processes in single cells. This approach overcomes the limitations involving heterogeneous cell populations and sample amounts, and may shed light on differential regulation of gene expression in normal versus disease-related contexts. Furthermore, high-throughput single-cell qRT-PCR provides a standardized, comparative assay for in-depth analysis of the mechanisms underlying human pluripotent stem cell self-renewal and differentiation.

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Figure 1: Schematic representation of the single-cell real-time PCR protocol.
Figure 2: Cell colony dissociation into single cells with Accutase.
Figure 3: Distribution in the 48 × 48 Dynamic Array.
Figure 4: Single-cell real-time PCR results.
Figure 5: Interpretation of heat map results.

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Acknowledgements

We are grateful to P.E. de Almeida for FACS discussion. We acknowledge funding support from the Swiss National Science Foundation PBBEP3_129803 (V.S.-F.); the German Research Foundation (A.D.E.); the Howard Hughes Medical Institute (S.R.Q.); the US National Institutes of Health (NIH) DP2OD004437, RC1AG036142, R01AI085575 (J.C.W.), and the Burroughs Wellcome Foundation (J.C.W.).

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V.S.-F. and A.D.E. prepared most of the paper. T.K., S.R.Q. and J.C.W. provided advice and proofread the paper.

Corresponding author

Correspondence to Joseph C Wu.

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Competing interests

Stephen R Quake is affiliated with Fluidigm. The rest of the authors declare no competing financial interests.

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Sanchez-Freire, V., Ebert, A., Kalisky, T. et al. Microfluidic single-cell real-time PCR for comparative analysis of gene expression patterns. Nat Protoc 7, 829–838 (2012). https://doi.org/10.1038/nprot.2012.021

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