HCR v3.0 Protocol Overview

HCR v3.0 enables multiplexed quantitative RNA fluorescence in situ hybridization (RNA-FISH), RNA flow cytometry, and northern blotting with automatic background suppression throughout the protocol. Protocols are simple, robust, and enzyme-free, requiring only 2 stages independent of the number of target RNAs.

Detection Stage

Detection Stage

An HCR probe set comprises multiple probe pairs that hybridize to different subsequences along the target with HCR initiator i1 split between each pair of probes. During the Detection Stage, probe pairs that hybridize specifically to the target RNA colocalize the full HCR initiator i1, while any individual probes that bind non-specifically to the sample do not colocalize the full HCR initiator i1.

Amplification Stage

Amplification Stage

An HCR amplifier comprises metastable HCR hairpins h1 and h2. Learn more

Specifically bound probe pairs colocalize the full HCR initiator i1 and trigger growth of a tethered fluorescent HCR amplification polymer. Non-specifically bound probes do not colocalize the full HCR initiator i1 and do not trigger HCR. Hairpins are kinetically trapped so any individual hairpins that bind non-specifically in the sample do not trigger HCR.

Automatic Background Suppression

HCR v3.0 reagents provide automatic background suppression throughout the protocol, ensuring that even if probes or hairpins bind non-specifically in the sample they will not generate amplified background, dramatically enhancing performance and ease-of-use. Learn more