qHCR imaging enables RNA relative quantitation with subcellular resolution in the anatomical context of thick autofluorescent samples.
Analog Quantitative Signal
qHCR signal is analog in the form of subcellular fluorescence voxel intensities that scale approximately linearly with the number of RNA target molecules per voxel.
Quantitative fluorescence in situ hybridization (FISH) in a whole-mount chicken embryo
Redundant 2-Channel Detection
To illustrate quantitative accuracy and precision with subcellular resolution within a whole-mount vertebrate embryo, we detect a target mRNA using two probe sets that trigger different HCR amplifiers carrying spectrally distinct fluorophores.
High Accuracy and Precision with Subcellular Resolution
In the resulting scatter plot of fluorescence voxel intensities, high accuracy corresponds to linearity with zero intercept, and high precision corresponds to tight scatter around the line. Voxels are subcellular (2×2×2 µm).
Subcellular fluorescence voxel intensities
Precision Increases with Voxel Size
Quantitative precision increases with voxel size (so long as voxels remain smaller than the expression domains under consideration). To maintain subcellular resolution while enhancing precision, we recommend averaging pixels to obtain roughly 2×2×2 µm voxels.
Precision Increases with Probe Set Size
Using HCR v3.0, automatic background suppression ensures that precision increases with probe set size. Learn more
As a result, for qHCR imaging, we recommend using 20 or more split-initiator probe pairs per target RNA (as permitted by target length).
Signal-to-Background Increases with Amplification Time
Signal increases with amplification time and background does not, so for qHCR imaging, we recommend overnight amplification to grow long amplification polymers and enhance the signal-to-background ratio.
Orthogonal HCR amplifiers enable 1-step signal amplification for up to 5 target RNAs simultaneously. Straightforward multiplexing is achieved using the same 1.5-day protocol independent of the number of target RNAs. Learn more
Custom Probe Set Design
Custom probe set design is available for any target mRNA in any organism across the tree of life. Learn more
Read-Out/Read-In Quantitative Discovery
By quantifying RNA within intact specimens, qHCR imaging enables bidirectional quantitative discovery in an anatomical context. Learn more
qHCR and dHCR Quantitative Imaging Modes
HCR v3.0 supports two quantitative imaging modes: analog RNA relative quantitation with subcellular resolution (qHCR mode) and digital RNA absolute quantitation with single-molecule resolution (dHCR mode). See comparison