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

Straightforward Multiplexing

Orthogonal HCR amplifiers enable 1-step signal amplification for up to 5 target RNAs simultaneously.  Learn more

Custom Probe Set Design

Custom probe set design is available for any target mRNA in any organism across the tree of life. Learn more

Quantitative Analysis Modes

Protocols are available for four quantitative analysis modes:

qHCR Imaging

Analog RNA relative quantitation with subcellular resolution in the anatomical context of thick autofluorescent samples. Learn more


dHCR Imaging

Digital RNA absolute quantitation with single-molecule resolution in the anatomical context of thick autofluorescent samples. Learn more

qHCR Flow Cytometry

Analog RNA relative quantitation for high-throughput expression profiling of mammalian and bacterial cells. Learn more


qHCR Northern Blots

Simultaneous quantification of RNA target size and abundance. Learn more

We recently updated our Privacy Policy to include new sections on Google Analytics and Do Not Track.

Please visit our Privacy Policy to see the full details.

Copyright © 2020 Molecular Instruments, Inc. All rights reserved.