HCR RNA-FISH: Protocol Overview
The same 2-stage protocol is used independent of the number of target RNAs.
All major
automation platforms
Straightforward Multiplexing
HCR™ enables straightforward multiplexing using 1-step quantitative signal amplification for all targets simultaneously.
Automatic Background Suppression
Because hairpins are kinetically trapped, they will not generate amplified background even if they bind non-specifically in the sample, providing automatic background suppression to dramatically enhance performance and ease-of-use.
Deep Sample Penetration
Amplification hairpins are short predominantly double-stranded oligos that penetrate thick samples to execute an enzyme-free amplification cascade at the site of the target.
Orthogonal Amplifiers
The gel above demonstrates multiplexed signal amplification by 4 orthogonal amplifiers (B1, B2, B3, B4). In the absence of initiators, there is negligible leakage out of the metastable hairpin state. Introduction of any single initiator (B1i1, B2i1, B3i1, B4i1) into the amplifier mixture triggers the corresponding amplifier.
Quantitation
The amplification mechanism is simple, robust, and enzyme-free, providing quantitative, high-gain, 1-step signal amplification for diverse analysis modes:
HCR™ Amplifier
HCR™ amplifiers enable multiplexed, quantitative, 1-step, isothermal, high-gain, enzyme-free signal amplification in diverse technological settings.
HCR™ Amplifier: How It Works
HCR™ Amplifier
HCR™ amplifiers introduced the concept of conditional nucleic acid self-assembly, demonstrating that nucleic acid molecules can be engineered so as not to interact when mixed, instead self-assembling only upon exposure to a cognate initiator sequence. An amplifier consists of two kinetically trapped hairpins, h1 and h2, that co-exist metastably on lab time scales. Hairpins h1 and h2 store the energy to drive a conditional self-assembly cascade upon exposure to initiator i1. Initiator i1 triggers a chain reaction of alternating h1 and h2 polymerization steps leading to growth of a fluorescent amplification polymer.
A new era for in situ hybridization:
multiplexed, quantitative, high-resolution, sensitive, versatile, robust
Experience multiplexed, quantitative, high-resolution immunfluorescence: amplified, enzyme-free, FFPE, simple
Quantitative 1-step enzyme-free HCR™ signal amplification for all protein and RNA targets simultaneously