HCR RNA-FISH: Protocol Overview
The same 2-stage protocol is used independent of the number of target RNAs.
All major
automation platforms
HCR™ Northern Blot Technology
Multiplexed quantitative RNA blotting
HCR™ probe sets, amplifiers, and buffers enable multiplexed quantification of RNA target size and abundance with automatic background suppression throughout the protocol.
Quantitative Signal
HCR™ signal is quantitative in the form of band intensities that scale approximately linearly with target abundance.
HCR™ Northern Blotting: How It Works
Detection Stage
Amplification Stage
Detection Stage
An HCR™ probe set comprises multiple probe pairs that hybridize to different subsequences along the target. Initiator i1 is split between each pair of probes such that only probe pairs that hybridize specifically to the target RNA colocalize the full initiator i1.
Amplification Stage
An HCR™ amplifier comprises metastable fluorescent hairpins h1 and h2. Specifically bound probe pairs that colocalize full initiator i1 trigger growth of a tethered fluorescent amplification polymer.
Automatic Background Suppression
HCR™ probes and amplifiers combine to provide automatic background suppression throughout the protocol: probes that bind non-specifically do not colocalize full initiator i1 and do not trigger amplification; hairpins that bind non-specifically are kinetically trapped and do not trigger amplification.
RNA Relative Quantitation via HCR™ Northern Blot
Relative Quantitation
For a given target, relative quantitation is performed by comparing band intensities between lanes.
3-Plex RNA Relative Quantitation
High Accuracy and Precision
Standard curves demonstrate high accuracy (linearity with zero intercept) and precision (tight scatter around the line).
RNA Absolute Quantitation via HCR™ Northern Blot
Absolute Quantitation
Absolute quantitation is performed by comparing samples to a standard curve generated using RNA targets of known abundance.
Absolute miRNA Quantitation for HEK 293T and HeLa Cells
High Accuracy and Precision
Standard curves demonstrate high accuracy (linearity with zero intercept) and precision (tight scatter around the line).
Simple Robust Protocols
Quantitative HCR™ northern blot protocols are simple, robust, and enzyme-free, requiring only 2 stages independent of the number of target RNAs.
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Automatic Background Suppression
HCR™ northern blot 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.
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Straightforward Multiplexing
HCR™ enables straightforward multiplexing using 1-step quantitative signal amplification for all target RNAs simultaneously.
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Custom Probe Design
Free custom probe design is available for any target mRNA in any organism across the tree of life.
Reagents will not generate amplified background even if they bind non-specifically within the sample
The same 2-stage enzyme-free protocol is used independent of the number of target RNAs
Custom probe set design for any target mRNA in any organism across the tree of life