HCR RNA-FISH: Protocol Overview
The same 2-stage protocol is used independent of the number of target RNAs.
Automatic Background Suppression
For multiplexed quantitative analysis of RNA targets using:
HCR probes and amplifiers combine to provide automatic background suppression throughout the protocol, ensuring that reagents will not generate amplified background even if they bind non-specifically within the sample.
Enhanced Performance and Ease-of-Use
Automatic background suppression dramatically enhances performance (signal-to-background and quantitative precision) and ease-of-use (no probe set optimization for new targets and organisms).
Automatic Background Suppression: How It Works
Detection Stage
Amplification Stage
HCR Split-Initiator Probe Pairs
An HCR probe set comprises multiple probe pairs that hybridize to different subsequences along the RNA 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. Probes that bind non-specifically do not colocalize full HCR initiator i1 and do not trigger HCR.
Kinetically Trapped HCR Hairpins
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. Hairpins that bind non-specifically are kinetically trapped and do not trigger HCR.
A new era for in situ hybridization:
multiplexed, quantitative, high-resolution, sensitive, versatile, robust
RNA relative quantitation for high-throughput expression profiling of mammalian cells and bacteria
Quantitative RNA Analysis Modes
Automatic background suppression enhances performance and ease-of-use for four quantitative RNA analysis modes:
qHCR RNA Imaging
qHCR RNA imaging enables analog RNA relative quantitation with subcellular resolution in the anatomical context of thick autofluorescent samples.
dHCR RNA Imaging
dHCR RNA imaging enables digital RNA absolute quantitation with single-molecule resolution in the anatomical context of thick autofluorescent samples.
qHCR RNA Flow Cytometry
qHCR RNA flow cytometry enables analog RNA relative quantitation for high-throughput expression profiling of mammalian cells and bacteria.
qHCR Northern Blots
qHCR northern blots enable simultaneous quantification of RNA target size and abundance.
Enhanced Performance
✓ Signal-to-background
✓ Quantitative precision