HCR RNA-FISH: Protocol Overview
The same 2-stage protocol is used independent of the number of target RNAs.
HCR RNA Flow Cytometry Technology
Multiplexed quantitative RNA flow cytometry
HCR probe sets, amplifiers, and buffers enable multiplexed quantitative RNA flow cytometry with automatic background suppression throughout the protocol, enabling high-throughput RNA expression profiling of mammalian cells and bacteria without the need to engineer reporter lines.
Quantitative Signal
qHCR signal is quantitative in the form of single-cell fluorescence intensities that scale approximately linearly with the number of RNA target molecules per cell.
HCR RNA Flow Cytometry: How It Works
Detection Stage
Amplification Stage
Detection Stage
An HCR probe set comprises multiple probe pairs that hybridize to different subsequences along the target. HCR initiator i1 is split between each pair of probes such that only probe pairs that hybridize specifically to the target RNA colocalize the full HCR initiator i1.
Amplification Stage
An HCR amplifier comprises metastable fluorescent HCR hairpins h1 and h2. Specifically bound probe pairs that colocalize full HCR initiator i1 trigger growth of a tethered fluorescent HCR amplification polymer.
Automatic Background Suppression
HCR probes and amplifiers combine to provide automatic background suppression throughout the protocol: probes that bind non-specifically do not colocalize full HCR initiator i1 and do not trigger HCR; hairpins that bind non-specifically are kinetically trapped and do not trigger HCR.
qHCR RNA Flow Cytometry in Mammalian Cells and Bacteria
Redundant 2-Channel Detection
To illustrate the quantitative nature of qHCR RNA flow cytometry, we detect a target mRNA using two probe sets that trigger different HCR amplifiers carrying spectrally distinct fluorophores.
Mammalian Cells (HEK 293T)
Bacteria (E. coli)
High Accuracy and Precision
The resulting scatter plots of single-cell HCR signal demonstrate high accuracy (linearity with zero intercept) and precision (tight scatter around the line) in mammalian cells and bacteria.
Simple Robust Protocols
HCR RNA flow cytometry protocols are simple, robust, and enzyme-free, requiring only 2 stages independent of the number of target RNAs.
Automatic Background Suppression
HCR v3.0 reagents provide automatic background suppression throughout the protocol, ensuring that even if probes or hairpins bind non-specifically in the sample they will not generate amplified background, dramatically enhancing performance and ease-of-use.
Straightforward Multiplexing
HCR enables straightforward multiplexing using 1-step quantitative signal amplification for all target RNAs simultaneously.
Custom Probe Design
Free custom probe design is available for any target mRNA in any organism across the tree of life.
Precision Increases with Probe Set Size
Quantitative precision increases with probe set size due to the benefits of automatic background suppression. For qHCR flow cytometry, we recommend using 30+ split-initiator probe pairs per target RNA (maximize probe set size given target length).
Signal-to-Background Increases with Amplification Time
For qHCR flow cytometry, we recommend overnight amplification to grow long amplification polymers that enhance the signal-to-background ratio.
Reagents will not generate amplified background even if they bind non-specifically within the sample
The same 2-stage enzyme-free protocol is used independent of the number of target RNAs
Custom probe set design for any target mRNA in any organism across the tree of life
qHCR RNA Flow Cytometry
✓ RNA relative quantitation
✓ High accuracy and precision
✓ High-throughput single-cell analysis
✓ Mammalian cells or bacteria
✓ Automatic background suppression throughout the protocol