启用 RNAscope ISH 技术

A New User's Guide to Getting Started with RNAscope ISH Technology

RNAscope ISH Technology is the gold standard for in situ hybridization (ISH), providing spatial visualization of gene expression with unparalleled sensitivity, specificity, and reliability.

This webinar will provide a comprehensive overview of our RNAscope ISH assays to help new RNAscope users set up and get started.

We  discuss the workflows for each manual assay, including required materials and reagents, tips for sample preparation and pre-treatment optimization, and guidelines for image acquisition and data analysis. We will also discuss recommendations for troubleshooting and optimization.

RNAscope ISH Pretreatment Reagents

RNAscope Pretreatment reagents provide the right tools for these important jobs. The proprietary formulations of these reagents have been optimized to provide enhanced access of in situ hybridization probes to nucleic acid targets across a wide variety of tissue sample types including FFPE, fresh‑frozen (FF), and fixed-frozen tissue as well as tissue microarray (TMA) and cultured adherent and non-adherent cells.

RNAscope Target Retrieval: A buffer system used along with heating of the samples to partially reverse the cross-linking that occurs from tissue fixation, including perfused tissue. This reagent is not required for fresh (unfixed) frozen tissue.

RNAscope Hydrogen Peroxide Reagent: For blocking endogenous peroxidase activity.

RNAscope Proteases: Reagent used to permeabilize cell membranes as well as to unmask RNA or DNA targets by degrading bound proteins. Different sample preparation methods may require reagents with different proteolytic activity. We offer several types of protease including RNAscope Protease Plus, Protease III, and Protease IV.

Pretreat Pro/AMP Pro: These reagents, in combination with retrieval and blocking where needed, provide a protease-free pretreatment workflow to streamline co-detection and multiomic workflows.

RNAscope Probe Hybridization Technology

RNAscope, BaseScope, and DNAscope assays employ a probe hybridization strategy that involves pairs of probes, each of which contains 18 to 25 bases complementary to a section of the target RNA adjacent to that of the other pair member. The overall system is designed such that both probes in each pair must hybridize independently to their respective target sequences for subsequent signal amplification to occur.

High Specificity, Excellent Signal-to-Noise Ratio

This pairing requirement not only has the effect of greatly increasing the signal-to-noise ratio but also ensures high specificity stemming from the extremely low probability of two different probes binding adjacent non-specific sequences. Furthermore, our probe design algorithm is validated to select for optimal hybridization within the recommended assay conditions and minimal cross-hybridization to off-target sequences.

Single RNA Molecule Detection Achieved with Degraded Samples or Less Accessible Targets

Although detection of a single RNA target molecule with an RNAscope assay can be accomplished by the binding of just three pairs of probes for the target, there are 20 separate pairs of probes specific to the target in the assay. This high level of redundancy means that even partially degraded or incompletely unmasked RNA targets may still be detectable at single-molecule resolution. Another feature that contributes to the robustness of RNAscope assays in this respect are the relatively short target regions required, with just 36 to 50 bases of combined target sequence being sufficient for the hybridization of a probe pair.

Adjacent Z-Probe Pairs Form a Binding Site for the Amplification Apparatus

The 18- to 25-base “lower” portion of each hybridization probe of the pair just discussed that binds specifically and adjacently to the target molecule is connected via a spacer sequence to a 14-base “upper” portion. The spacer sequences cause each probe construct to assume a “Z” shape wherein the two upper portions of a bound pair will also lie adjacently in a line. This upper structure forms a 28-base binding site for a so-called pre-amplifier molecule.

As additional insurance against signals arising from non-specific binding, the pre-amplifier requires both upper portions, i.e., the entire 28-base sequence, to be presented contiguously to bind.

Many Amplifiers Bind Each Pre-Amplifier; Each Amplifier Binds Many Labeled Probes

Once bound to the upper portion of the probe pair, the pre-amplifier provides a kind of scaffolding that bears numerous binding sites for “amplifier” molecules, each of which contains a similar number of binding sites for labeled probes that contain either a chromogenic enzyme or a fluorescent molecule.

The multiplicity of labeled probes arising from the binding of a single molecule is what enables the dramatic levels of signal amplification characteristic of RNAscope assays.

Detection by Microscopy

Detection is accomplished via fluorescent or brightfield microscopic analysis, depending on the type of assay selected. Each punctate dot signal represents a single target molecule. The system can also be automated on partner platforms (Leica Biosystems and Roche Tissue Diagnostics). Single-molecule signals can be quantified on a cell-by-cell basis by manual counting or automated using commercial image processing software packages. We provide technical notes for the open-source image processing programs QuPath, ImageJ, and CellProfiler.