Citation Notes

For citation, please select from the list below as appropriate for your application:


  • HCR v3.0
    Automatic background suppression 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:


qHCR imaging: analog mRNA relative quantitation with subcellular resolution in the anatomical context of thick autofluorescent samples. 

qHCR flow cytometry: analog mRNA relative quantitation for high-throughput expression profiling of mammalian and bacterial cells.
dHCR imaging: digital mRNA absolute quantitation with single-molecule resolution in the anatomical context of thick autofluorescent samples.

Protocols for v3.0 in diverse organisms are adapted from the Zoo paper.

  • qHCR imaging
    mRNA relative quantitation with subcellular resolution in the anatomical context of whole-mount vertebrate embryos; read-out/read-in analysis framework (Trivedi et al., 2018). 


  • Zoo paper
    Protocols for multiplexed mRNA imaging in diverse sample types (Choi et al., 2016):​

bacteria in suspension

FFPE human tissue sections 

generic sample in solution

generic sample on a slide

mammalian cells on a slide

mammalian cells in suspension

whole-mount chicken embryos

whole-mount fruit fly embryos  

whole-mount mouse embryos

whole-mount nematode larvae

whole-mount sea urchin embryos

whole-mount zebrafish embryos and larvae

  • dHCR imaging
    Single-molecule mRNA imaging in thick autofluorescent samples (e.g., 0.5 mm adult mouse brain sections) (Shah et al., 2016). 


  • qHCR northern blot
    Simultaneous quantification of RNA target size and abundance for up to 5 target RNAs (Schwarzkopf & Pierce, 2016). 


  • HCR v2.0
    2nd generation in situ HCR 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). 


  • HCR v1.0
    1st generation in situ HCR 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). ​

  • HCR mechanism
    Isothermal enzyme-free molecular signal amplification (Dirks & Pierce, 2004). ​​


  • 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. 

  • 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. 

  • 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. 

  • Dirks, R.M., & Pierce, N.A. (2004). Triggered amplification by hybridization chain reaction. Proc Natl Acad Sci USA, 101(43), 15275–15278. 

  • Schwarzkopf, M., & Pierce, N.A. (2016). Multiplexed miRNA northern blots via hybridization chain reaction. Nucleic Acids Res, 44(15), e129. 

  • 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. 

  • 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.