Property

Signal

 

Probe set size

Amplification time

Magnification

Target expression level

Comparison of qHCR and dHCR Quantitative Imaging Modes

HCR v3.0 reagents support two quantitative imaging modes:

qHCR Imaging

Analog RNA relative quantitation with subcellular resolution in the anatomical context of thick autofluorescent samples. Learn more

dHCR Imaging

Digital RNA absolute quantitation with single-molecule resolution in the anatomical context of thick autofluorescent samples. Learn more

Same Reagents For Both Modes

The same HCR probes, amplifiers, and buffers can be used in either imaging mode, so imaging can be performed in qHCR mode (longer amplification time, lower magnification) or dHCR imaging mode (shorter amplification time, higher magnification) depending on the expression level observed in situ. 

dHCR imaging

Digital single-molecule dots

30+ split-initiator probe pairs

45-90 min

Higher (63× to 100×)

Low or medium

qHCR Imaging

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

​Limiting Behavior of Signal 

Because the qHCR 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 dHCR signal per target molecule is digital, it remains the same as the number of target molecules decreases to zero. 

​Complementary Imaging Modes​​

Hence, qHCR and dHCR quantitative imaging modes are complementary, with qHCR suitable for medium- and high-copy targets (where the quantitative signal dominates autofluorescent background) and dHCR suitable for low-copy and medium-copy targets (where the signal from individual target molecules can be spatially separated)

Comparison to qPCR and dPCR

Just as quantitative PCR (qPCR) enables analog RNA relative quantitation in vitro, qHCR imaging enables analog RNA relative quantitation in situ. Just as digital PCR (dPCR) enables digital RNA absolute quantitation in vitro, dHCR imaging enables digital RNA absolute quantitation in situ. 

Read-Out/Read-In Quantitative Discovery

By quantifying RNA within intact specimens, qHCR and dHCR imaging modes both enable bidirectional quantitative discovery moving back and forth between expression space and anatomical space. Learn more

qHCR and dHCR Imaging Modes
 HCR v3.0 probe sets, amplifiers, and buffers
✓ RNA quantitation in an anatomical context

✓ Analog vs digital signal

Relative vs absolute quantification

✓ Subcellular vs single-molecule resolution

Thick autofluorescent samples

Automatic background suppression

We recently updated our Privacy Policy to include new sections on Google Analytics and Do Not Track.

Please visit our Privacy Policy to see the full details.

Copyright © 2019 Molecular Instruments, Inc. All rights reserved.