HCR™ Technology Citation Notes
For citation, please select from the list below as appropriate for your application:
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HCR™ IF + HCR™ RNA-FISH
HCR™ IF + HCR™ RNA-FISH enables a unified approach to multiplexed, quantitative, high-resolution protein immunofluorescence (IF) and RNA fluorescence in situ hybridization (RNA-FISH), with quantitative 1-step enzyme-free HCR™ signal amplification performed for all protein and RNA targets simultaneously (Schwarzkopf et al., 2021).
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HCR™ IF
HCR™ IF enables multiplexed, quantitative, high-resolution protein immunofluorescence (IF) in highly autofluorescent samples (e.g., FFPE brain tissue sections) (Schwarzkopf et al., 2021).
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HCR™ RNA-FISH (v3.0)
Third-generation HCR™ RNA-FISH (v3.0) enables multiplexed, quantitative, high-resolution RNA fluorescence in situ hybridization (RNA-FISH) with automatic background suppression throughout the protocol for dramatically enhanced performance (signal-to-background, qHCR™ precision, dHCR™ fidelity) and ease-of-use (no probe set optimization for new targets and organisms) (Choi et al., 2018). Quantitative analysis modes:-
Subcellular HCR™ RNA-FISH: analog mRNA relative quantitation with subcellular resolution in the anatomical context of thick autofluorescent samples.
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Single-molecule HCR™ RNA-FISH: digital mRNA absolute quantitation with single-molecule resolution in the anatomical context of thick autofluorescent samples.
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Quantitative HCR™ RNA flow cytometry: analog mRNA relative quantitation for high-throughput expression profiling of mammalian cells and bacteria.
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Protocols for HCR™ RNA-FISH (v3.0) in diverse organisms are adapted from the Zoo paper.
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Subcellular Quantitative RNA Imaging
Subcellular HCR™ RNA-FISH enables mRNA relative quantitation with subcellular resolution in the anatomical context of thick autofluorescent samples (e.g., whole-mount vertebrate embryos). The read-out/read-in analysis framework enables bidirectional quantitative discovery in an anatomical context (Trivedi et al., 2018).
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HCR™ RNA-FISH zoo
Protocols for multiplexed mRNA imaging in diverse sample types (Choi et al., 2016):-
bacteria in suspension
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FFPE human tissue sections
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generic sample in solution
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generic sample on a slide
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mammalian cells on a slide
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mammalian cells in suspension
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whole-mount chicken embryos
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whole-mount fruit fly embryos
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whole-mount mouse embryos
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whole-mount nematode larvae
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whole-mount sea urchin embryos
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whole-mount zebrafish embryos and larvae
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Single-molecule quantitative RNA imaging
Single-molecule HCR™ RNA-FISH enables RNA absolute quantitation with single-molecule resolution in the anatomical context of thick autofluorescent samples (e.g., 0.5 mm adult mouse brain sections) (Shah et al., 2016).
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Quantitative HCR™ northern blots
Quantitative HCR™ northern blots enable multiplexed quantification of RNA target size and abundance for up to 5 target RNAs (Schwarzkopf & Pierce, 2016).
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HCR™ RNA-FISH (v2.0)
Second-generation HCR™ RNA-FISH technology (v2.0) using DNA HCR™ probes and DNA HCR™ amplifiers: 10× increase in signal, 10× reduction in cost, dramatic increase in reagent durability (Choi et al., 2014).
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HCR™ RNA-FISH (v1.0)
First-generation HCR™ RNA-FISH technology (v1.0) using RNA HCR™ probes and RNA HCR™ amplifiers: multiplexed mRNA imaging in whole-mount vertebrate embryos with simultaneous signal amplification for up to 5 target mRNAs (Choi et al., 2010).
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HCR™ amplifiers
HCR™ amplifiers enables multiplexed, quantitative, 1-step, isothermal, enzyme-free signal amplification in diverse technological settings (Dirks & Pierce, 2004).
HCR™ Technology References
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Choi, H.M.T., Chang, J.Y., Trinh, L.A., Padilla, J.E., Fraser, S.E., & Pierce, N.A. (2010). Programmable in situ amplification for multiplexed imaging of mRNA expression.
Nat Biotechnol, 28:1208–1212. -
Choi, H.M.T., Beck, V.A., & Pierce, N.A. (2014). Next-generation in situ hybridization chain reaction: higher gain, lower cost, greater durability. ACS Nano, 8(5):4284-4294.
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Choi, H.M.T., Calvert, C.R., Husain, N., Huss, D., Barsi, J.C., Deverman, B.E., Hunter, R.C., Kato, M., Lee, S.M., Abelin, A.C.T., Rosenthal, A.Z., Akbari, O.S., Li, Y., Hay, B.A., Sternberg, P.W., Patterson, P.H., Davidson, E.H., Mazmanian, S.K., Prober, D.A., van de Rijn, M., Leadbetter, J.R., Newman, D.K., Readhead, C., Bronner, M.E., Wold, B., Lansford, R., Sauka-Spengler, T., Fraser, S.E., & Pierce, N.A. (2016). Mapping a multiplexed zoo of mRNA expression. Development, 143:3632-3637.
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Choi, H.M.T., Schwarzkopf, M., Fornace, M.E., Acharya, A., Artavanis, G., Stegmaier, J., Cunha, A., & Pierce, N.A. (2018). Third-generation in situ hybridization chain reaction: multiplexed, quantitative, sensitive, versatile, robust. Development, 145, dev165753.
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Dirks, R.M., & Pierce, N.A. (2004). Triggered amplification by hybridization chain reaction. Proc Natl Acad Sci USA, 101(43), 15275–15278.
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Schwarzkopf, M., & Pierce, N.A. (2016). Multiplexed miRNA northern blots via hybridization chain reaction. Nucleic Acids Res, 44(15), e129.
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Schwarzkopf, M., Liu, M.C., Schulte, S.J., Ives, R., Husain, N., Choi, H.M.T., & Pierce, N.A. (2021). Hybridization chain reaction enables a unified approach to multiplexed, quantitative, high-resolution immunohistochemistry and in situ hybridization. Development, 148(22):dev199847.
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Shah, S., Lubeck, E., Schwarzkopf, M., He, T.-F., Greenbaum, A., Sohn, C.H., Lignell, A., Choi, H.M.T., Gradinaru, V., Pierce, N.A., & Cai, L. (2016). Single-molecule RNA detection at depth via hybridization chain reaction and tissue hydrogel embedding and clearing. Development, 143:2862-2867.
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Trivedi, V., Choi, H.M.T., Fraser, S.E., & Pierce, N.A. (2018). Multidimensional quantitative analysis of mRNA expression within intact vertebrate embryos. Development, 145, dev156869.