Single-Molecule Quantitative RNA Imaging

Single-Molecule Fluorescence In Situ Hybridization (smFISH)

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.

Quantitative Signal

Single-molecule 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

High-Fidelity Single-Molecule RNA Imaging

HCR™ RNA-FISH 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 single-molecule HCR™ RNA-FISH, we detect a target mRNA using two probe sets that trigger different amplifiers carrying spectrally distinct fluorophores.

qHCR-imaging_qHCRimaging-RNA-redundant-d

Single-Molecule RNA Imaging Pointers

Fidelity Increases with Probe Set Size

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

Single-Molecule Resolution and Sensitivity

For single-molecule HCR™ RNA-FISH, we recommend 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™ RNA-FISH protocols are compatible with tissue clearing to reduce background in highly autofluorescent tissues.

Single-Molecule and Subcellular Quantitative Imaging Modes

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

Comparison of Single-Molecule HCR™ RNA-FISH to dPCR

While digital PCR (dPCR) enables digital RNA absolute quantitation in vitro without anatomical context, single-molecule HCR™ RNA-FISH enables digital RNA absolute quantitation in situ with anatomical context.