HCR RNA-FISH: Protocol Overview
The same 2-stage protocol is used independent of the number of target RNAs.
All major
automation platforms
HCR™ RNA-FISH Technology
Multiplexed, quantitative, high-resolution RNA imaging
HCR™ probe sets, amplifiers, and buffers enable multiplexed, quantitative, high-resolution RNA fluorescence in situ hybridization (RNA-FISH) with automatic background suppression throughout the protocol.
HCR™ RNA-FISH: 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.
Simple Robust Protocols
HCR™ RNA-FISH protocols are simple, robust, and enzyme-free, requiring only 2 stages independent of the number of target RNAs.
Automatic Background Suppression
HCR™ RNA-FISH 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.
Straightforward Multiplexing
HCR™ enables straightforward multiplexing using 1-step quantitative signal amplification for all target RNAs simultaneously.
Deep Sample Penetration
HCR™ amplification hairpins are short predominantly double-stranded oligos that penetrate thick samples to execute an enzyme-free amplification cascade at the site of the target.
Image Any Target mRNA. In Any Organism. In Any Sample Type.
HCR™ offers unmatched versatility (see gallery): FFPE tissue sections, fresh/fixed frozen tissue sections, thick brain slices, cells in solution or on a slide, whole-mounts, cleared samples, model organisms, non-model organisms, environmental samples, or multi-kingdom samples.
Custom Probe Design
Free custom probe design is available for any target mRNA in any organism across the tree of life.
HCR™ IF + HCR™ RNA-FISH
Combine HCR™ IF probes with HCR™ RNA-FISH probes to enable HCR™ protein-IF + HCR™ RNA-FISH for multiplexed, quantitative, high-resolution protein and RNA imaging with 1-step HCR™ signal amplification for all protein and RNA targets simultaneously.
Quantitative High-Resolution RNA Imaging
HCR™ RNA-FISH enables quantification of RNA targets with subcellular and single-molecule resolution in an anatomical context using two complementary imaging modes (see subcellular/single-molecule comparison):
Subcellular Quantitative RNA Imaging
Subcellular RNA imaging enables analog RNA relative quantitation with subcellular resolution in the anatomical context of thick autofluorescent samples.
Single-molecule Quantitative RNA Imaging
Single-molecule RNA imaging enables digital RNA absolute quantitation with single-molecule resolution in the anatomical context of thick autofluorescent samples.
Compatible with Tissue Clearing
HCR™ RNA-FISH protocols are compatible with tissue clearing to reduce background in highly autofluorescent tissues.
Reagents will not generate amplified background even if they bind non-specifically within the sample
Analog RNA relative quantitation with subcellular resolution in the anatomical context of thick autofluorescent samples
The same 2-stage enzyme-free protocol is used independent of the number of target RNAs
Digital RNA absolute quantitation with single-molecule resolution in the anatomical context of thick autofluorescent samples
Custom probe set design for any target mRNA in any organism across the tree of life
