Third-generation in situ hybridization chain reaction: multiplexed, quantitative, sensitive, versatile, robust

In situ hybridization based on the hybridization chain reaction (HCR) process has solved multi-decade challenges that hampered imaging of mRNA expression in diverse species, providing a rare combination of multiplexing, quantitation, specificity, resolution, and flexibility. It was complemented these capabilities with third-generation in situ HCR probes and amplifiers that work together to provide automated background suppression throughout the protocol, ensuring that reagents do not produce amplified background even though they bind non-specifically throughout the sample. Automatic background suppression improves accuracy and robustness by balancing the advantages of a higher signal-to-background ratio with the ease of using unoptimized probe sets for new targets and species.

In situ HCR v3.0 enables three multiplexed quantitative analysis modes:

1. qHCR imaging – analog mRNA relative quantitation with subcellular resolution in the anatomical context of whole-mount vertebrate embryos
2. qHCR flow cytometry – analog mRNA relative quantitation for high-throughput expression profiling of mammalian and bacterial cells
3. dHCR imaging – digital mRNA absolute quantitation via single-molecule imaging in thick autofluorescent samples

How does it work?

In this figure, you see HCR mechanism. Green stars denote fluorophores. Arrowhead indicates 3′ end of each strand. Here is the steps as following;

(A) In vitro, in situ, and in vivo, HCR produces isothermal enzyme-free signal amplification in a variety of technical environments. Each HCR amplification is made up of two types of kinetically trapped DNA hairpins (H1 and H2) that co-exist metastably on lab time scales, storing the energy to drive a conditional self-assembly cascade when exposed to a cognate DNA initiator chain (I1). Initiator I1 hybridizes to the input domain of hairpin H1, revealing its output domain, which in turn hybridizes to the input domain of hairpin H2, exposing its output domain, which is similar in sequence to initiator I1, forming the basis for a chain reaction of alternating H1 and H2 polymerization steps.

(B) Standard probes carry full HCR initiator I1 and generate amplified background if they bind non-specifically. Split-initiator probes P1 and P2 each carry half of HCR initiator I1 and do not generate amplified background if they bind non-specifically.

(C) Two-stage in situ HCR protocol. Detection stage: probe sets hybridize to mRNA targets, unused probes are washed from the sample. Amplification stage: specifically bound probe pairs trigger self-assembly of a tethered fluorescent amplification polymer and unused hairpins are washed from the sample. Automatic background suppression throughout the protocol: any reagents that bind non-specifically do not lead to generation of amplified background.

(D)Multiplexing timeline. The same two-stage protocol is used independent of the number of target mRNAs. HCR amplification is performed overnight for qHCR imaging and qHCR flow cytometry experiments (to maximize the signal-to-background ratio) and for 45-90 min for dHCR imaging experiments (to resolve individual molecules as diffraction-limited dots).

Check this paper for details of the protocol.