Subcellular Quantitative RNA and Protein Imaging

Multiplex, quantitative, high-resolution RNA-FISH, IF, and RNA-FISH/IF

HCR™ Gold RNA-FISH, HCR™ Gold IF, and HCR™ Gold RNA-FISH/IF enable simultaneous RNA and protein relative quantitation with subcellular resolution in the anatomical context of highly autofluorescent samples

Subcellular Quantitative Signal

Subcellular signal provides analog quantitation in the form of fluorescence voxel intensities that scale approximately linearly with the number of protein or RNA target molecules per subcellular voxel

Subcellular Quantitative RNA Imaging in a Whole-Mount Chicken Embryo

Redundant 2-Channel Detection

To illustrate the quantitative nature of subcellular HCR™ RNA-FISH, we detect a target mRNA using two probe sets that trigger different amplifiers carrying spectrally distinct fluorophores.

Fluorescence voxel intensities

High Accuracy and Precision with Subcellular Resolution

The resulting scatter plot of fluorescence voxel intensities demonstrates high accuracy (linearity with zero intercept) and precision (tight scatter around the line) for subcellular voxels (2.1×2.1×2.7 µm) within a whole-mount vertebrate embryo.

Subcellular Quantitative Protein Imaging in FFPE Human Breast Tissue Sections

Redundant 2-Channel Detection

To illustrate the quantitative nature of subcellular HCR™ IF, we detect a target protein using an unmodified 1º antibody probe and two batches of 2º antibody probes that trigger different amplifiers carrying spectrally distinct fluorophores.

Fluorescence voxel intensities

High Accuracy and Precision with Subcellular Resolution

The resulting scatter plot of fluorescence voxel intensities demonstrates high accuracy (linearity with zero intercept) and precision (tight scatter around the line) for subcellular voxels (2.0×2.0×2.5 µm) within an FFPE human breast tissue section.

Subcellular Quantitative Imaging Pointers

Precision Increases with Voxel Size

Quantitative precision increases with voxel size (so long as voxels remain smaller than the expression domains under consideration). To achieve high precision with subcellular resolution we recommend averaging pixels to obtain roughly 2×2×2 µm voxels.

Precision Increases with Number of Binding Sites for RNA Targets

Imaging RNA targets using HCR™ Gold RNA-FISH, precision increases with number of target-binding sites due to the benefits automatic background suppression. For subcellular quantitative HCR™ Gold RNA-FISH, we recommend using 20+ binding sites per target RNA (as permitted by target length).

Signal-to-Background Increases with Amplification Time

For subcellular protein and/or RNA imaging, we recommend overnight amplification to grow long amplification polymers that enhance the signal-to-background ratio.

Comparison of Subcellular HCR™ Gold to qPCR

While quantitative PCR (qPCR) enables analog RNA relative quantitation in vitro without anatomical context, subcellular HCR™ Gold RNA-FISH/IF enables analog protein and/or RNA relative quantitation in situ with anatomical context.