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.
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.
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
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:
Analog RNA relative quantitation with subcellular resolution in the anatomical context of thick autofluorescent samples. Learn more
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
Download HCR v3.0 Protocols
qHCR and dHCR Imaging Protocols
qHCR Flow Cytometry Protocols
qHCR Northern Blot Protocol