dHCR RNA Imaging

Single-Molecule Fluorescence In Situ Hybridization (smFISH) 

dHCR RNA imaging using HCR RNA-FISH enables RNA absolute quantitation with single-molecule resolution in the anatomical context of thick autofluorescent samples, providing automatic background suppression throughout the protocol for dramatically enhanced performance and ease-of-use.

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Quantitative Signal

dHCR signal provides digital quantitation in the form of diffraction-limited dots representing individual RNA target molecules. 

High-Fidelity smFISH in a Whole-Mount Chicken Embryo
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High-Fidelity Single-Molecule RNA Imaging

dHCR imaging enables high-fidelity imaging of single mRNA molecules even in thick autofluorescent samples (e.g., whole-mount vertebrate embryos and thick brain slices). 

Redundant 2-Channel Detection

To illustrate the quantitative nature of dHCR imaging, we detect a target mRNA using two probe sets that trigger different HCR amplifiers carrying spectrally distinct fluorophores.  

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dHCR Imaging Pointers

Fidelity Increases with Probe Set Size

Fidelity increases with probe set size due to the benefits of automatic background suppression. For dHCR RNA imaging, we recommend using 30+ split-initiator probe pairs per target RNA (maximize probe set size given target length). 

Single-Molecule Resolution and Sensitivity

For dHCR imaging, we recommend HCR signal amplification for 45-90 minutes to grow amplification polymers that are short enough to retain diffraction-limited single-molecule resolution, but long enough to provide single-molecule sensitivity even in thick autofluorescent samples (e.g., thick brain slices). 

Compatible with Tissue Clearing

HCR imaging protocols are compatible with tissue clearing to reduce background in highly autofluorescent tissues. 

dHCR and qHCR Quantitative Imaging Modes 

HCR RNA-FISH supports two quantitative RNA imaging modes (see comparison): dHCR imaging (digital RNA absolute quantitation with single-molecule resolution) and qHCR imaging (analog RNA relative quantitation with subcellular resolution). 

Comparison of dHCR to dPCR

While digital PCR (dPCR) enables digital RNA absolute quantitation in vitro without anatomical context, dHCR imaging enables digital RNA absolute quantitation in situ with anatomical context. 

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Reagents will not generate amplified background even if they bind non-specifically within the sample

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The same 2-stage enzyme-free protocol is used independent of the number of target RNAs

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Custom probe set design for any target mRNA in any organism across the tree of life

dHCR Imaging

 Digital RNA absolute quantitation

✓ Single-molecule resolution and sensitivity

✓ Thick autofluorescent samples

✓ Compatible with tissue clearing

✓ Automatic background suppression throughout the protocol