Automatic Background Suppression

HCR v3.0 uses probes and amplifiers that 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. 

Stage 1: Detection

Split-Initiator HCR Probes

HCR initiator i1 is split between a pair of probes so that any individual probes that bind non-specifically in the sample will not trigger HCR.  The target RNA mediates signal amplification by recruiting both probes within a pair to adjacent binding sites, colocalizing a full HCR initiator i1 capable of triggering HCR. 

Stage 2: Amplification

Kinetically Trapped HCR Hairpins

Fluorophore-labeled HCR hairpins h1 and h2 are kinetically trapped so that any individual hairpins that bind non-specifically in the sample will not polymerize. On the other hand, each full HCR initiator i1 colocalized by the target RNA will trigger growth of a tethered fluorescent HCR amplification polymer. 

Enhanced Performance and Ease-of-Use

Automatic background suppression throughout the protocol dramatically enhances performance and ease-of-use across four quantitative analysis modes: 

qHCR Imaging

Automatic background suppression enhances signal-to-background and quantitative precision for qHCR imaging, enabling RNA quantification with subcellular resolution in thick autofluorescent samples (e.g., whole-mount vertebrate embryos). Learn more

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dHCR Imaging

Automatic background suppression enhances fidelity for dHCR imaging, enabling single-molecule imaging even in thick autofluorescent samples (e.g., thick brain slices). Learn more

qHCR Flow Cytometry

Automatic background suppression enhances signal-to-background and precision for qHCR flow cytometry, enabling RNA expression profiling in both mammalian and bacterial cells. Learn more

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qHCR Northern Blots

Automatic background suppression enhances signal-to-background and precision for quantification of RNA target abundance using qHCR northern blots. Learn more