HCR RNA-FISH: Protocol Overview
The same 2-stage protocol is used independent of the number of target RNAs.
Property
Signal
Probe set size
Amplification time
Magnification
Target expression level
Property
Signal
Probe set size
Amplification time
Magnification
Target expression level
Comparison of Subcellular and Single-Molecule Quantitative RNA Imaging Modes
HCR™ RNA-FISH probe sets, amplifiers, and buffers support two quantitative imaging modes with automatic background suppression throughout the protocol:
Subcellular Quantitative RNA Imaging
Subcellular HCR™ RNA-FISH enables analog RNA relative quantitation with subcellular resolution in the anatomical context of thick autofluorescent samples.
Single-Molecule Quantitative RNA Imaging
Single-molecule HCR™ RNA-FISH enables digital RNA absolute quantitation with single-molecule resolution in the anatomical context of thick autofluorescent samples.
Single-Molecule Imaging Mode
Digital single-molecule dots
30+ split-initiator probe pairs
45-90 min
Higher (63× to 100×)
Low or medium
Subcellular Imaging Mode
Analog subcellular voxel intensities
20+ split-initiator probe pairs
Overnight
Lower (20× to 40×)
Medium or high
Property
Signal
Probe set size
Amplification time
Magnification
Target expression level
Single-molecule RNA imaging
Digital single-molecule dots
30+ split-initiator probe pairs
45-90 min
Higher (63× to 100×)
Low or medium
Subcellular RNA Imaging
Analog subcellular voxel intensities
20+ split-initiator probe pairs
Overnight
Lower (20× to 40×)
Medium or high
Same Reagents For Both Imaging Modes
The same HCR™ probes, amplifiers, and buffers are used for both imaging modes, so imaging can be performed in subcellular mode (longer amplification time, lower magnification) or single-molecule imaging mode (shorter amplification time, higher magnification) depending on the expression level observed in situ.
​Limiting Signal Behavior
Because the subcellular signal per imaging voxel is analog, it will properly decrease to zero as the number of targets per imaging voxel decreases to zero. However, because the single-molecule signal per target molecule is digital, it remains the same as the number of target molecules decreases to zero. ​
Complementary Imaging Modes​​
Subcellular and single-molecule quantitative imaging modes are complementary, with subcellular suitable for medium- and high-copy targets (where the quantitative signal dominates autofluorescent background) and single-molecule suitable for low-copy and medium-copy targets (where the signal from individual target molecules can be spatially separated).
Comparison of subcellular HCR™ RNA-FISH to qPCR
While quantitative PCR (qPCR) enables analog RNA relative quantitation in vitro without anatomical context, subcellular HCR™ RNA-FISH enables analog RNA relative quantitation in situ with anatomical context.
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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.
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Read-Out/Read-In Quantitative Discovery
By quantifying RNA while preserving the sample anatomy, subcellular and single-molecule HCR™ RNA-FISH enable bidirectional quantitative discovery using read-out/read-in analyses to move back and forth between expression space and anatomical space.
Subcellular and Single-Molecule Quantitative RNA Imaging Modes
✓ HCR™ RNA-FISH probe sets, amplifiers, and buffers
✓ RNA quantitation in an anatomical context
✓ Analog or digital signal
✓ Relative or absolute quantification
✓ Subcellular or single-molecule resolution
✓ Thick autofluorescent samples
✓ Automatic background suppression throughout the protocol
All major
automation platforms
