Abstract
In situ hybridization based on the mechanism of hybridization chain reaction (HCR) enables high-throughput expression profiling of mammalian or bacterial cells via flow cytometry. Third-generation in situ HCR (v3.0) provides automatic background suppression throughout the protocol, dramatically enhancing performance and ease of use. In situ HCR v3.0 supports analog mRNA relative quantitation via qHCR flow cytometry. Here, we provide protocols for multiplexed qHCR flow cytometry for mammalian or bacterial cells in suspension.
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References
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Acknowledgments
We thank A. Acharya and G. Artavanis for performing preliminary studies. Within the Beckman Institute at Caltech, we thank the following for assistance: C.R. Calvert and G.J. Shin (Molecular Technologies), R. Diamond, and D. Perez (Flow Cytometry Facility). This work was funded by the National Institutes of Health (National Institute of Biomedical Imaging and Bioengineering R01EB006192), by the Defense Advanced Research Projects Agency (HR0011-17-2-0008; the findings are those of the authors and should not be interpreted as representing the official views or policies of the US Government), by the Beckman Institute at Caltech (Programmable Molecular Technology Center, PMTC), by the Gordon and Betty Moore Foundation (GBMF2809), by the National Science Foundation Molecular Programming Project (NSF-CCF-1317694), by a Professorial Fellowship at Balliol College, University of Oxford, and by the Eastman Visiting Professorship at the University of Oxford.
Competing Interests
The authors declare competing financial interests in the form of patents, pending patent applications, and a startup company (Molecular Instruments).
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Schwarzkopf, M., Choi, H.M.T., Pierce, N.A. (2020). Multiplexed Quantitative In Situ Hybridization for Mammalian or Bacterial Cells in Suspension: qHCR Flow Cytometry (v3.0). In: Nielsen, B.S., Jones, J. (eds) In Situ Hybridization Protocols . Methods in Molecular Biology, vol 2148. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0623-0_8
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DOI: https://doi.org/10.1007/978-1-0716-0623-0_8
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Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-0622-3
Online ISBN: 978-1-0716-0623-0
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