How Spatial Genomic ISH Works

Leading the field in spatial genomics, RNAscope Technology is a novel in situ hybridization (ISH) assay for detection of target RNA within intact cells. The assay represents a major advance in RNA ISH approaches with its proprietary probe design to amplify target-specific signals but not background noise from non-specific hybridization.

RNAscope™ In Situ Hybridization Assay Workflow

  • Step 1:


    Tissue sections or cells are fixed onto slides and pretreated with RNAscope™ Pretreatment Kit to unmask target RNA and permeabilize cells.

  • Step 2:


    Designed with ~20 target-specific double Z probes, RNAscope™ Probes hybridize to target RNA molecules.

  • Step 3:


    RNAscope™ Detection Reagents amplify the hybridization signals via sequential hybridization of amplifiers and label probes.

  • Step 4:


    Each punctate dot signal represents a single test target RNA molecule and can be visualized with a microscope.

  • Step 5:


    Single-molecule signals can be quantified on a cell-by-cell basis by manual counting or automated image analysis using HALO Software.

RNAscopeTM Probe Design and Signal Amplification Strategy

In order to substantially improve the signal-to-noise ratio of RNA ISH, RNAscope employs a probe design strategy much akin to fluorescence resonance energy transfer (FRET), in which two independent probes (double Z probes) have to hybridize to the target sequence in tandem in order for signal amplification to occur. As it is highly unlikely that two independent probes will hybridize to a non-specific target right next to each other, this design concept ensures selective amplification of target-specific signals.

For each target RNA species, ~20 double Z target probe pairs are designed to specifically hybridize to the target molecule, but not to non-targeted molecules.

Each Target Z Probe Contains Three Elements

The lower region of the Z is an 18-to 25-base region that is complementary to the target RNA. This sequence is selected for target specific hybridization and uniform hybridization properties.

A spacer sequence that links the two components of the probe. The upper region of the Z is a 14-base tail sequence.

The two tails from a double Z probe pair forms a 28 base binding site for the pre-amplifier.

Signal Amplification is Achieved by a Cascade of Hybridization Events

Step 1. Double Z target probes hybridize to the RNA target (~1kb)

Step 2. Pre-amplifiers hybridize to the 28-base binding site formed by each double Z probe

Step 3: Amplifiers are then binding to the multiple binding sites on each preamplifier.

Step 4: Labeled probes, containing a fluorescent molecule or chromogenic enzyme, bind to the numerous binding sites on each amplifier.

Advantages of RNAscope Probe Design and Signal Amplification Strategy

Detection of each single RNA molecule requires only three double Z probes to bind to target RNA.

The 20 double Z probes provide robustness against partial target RNA accessibility or degradation.


The double Z probe design prevents background noise. Single Z probes binding to nonspecific site will not produce a binding site for the pre-amplifier, thus preventing amplification of non-specific signals, contributing to specificity.

Single Molecule Detection

The 20x20x20 probe design and signal amplification increases sensitivity such that a single molecule of RNA can be visualize as a punctuate signal dot under a standard microscope.

Degraded Sample Compatible

The double Z probe design, with its relatively short target region (40-50 bases of the lower region of the double Z) allows for successful hybridization of partially degraded RNA.

This publication details the core principles behind RNAscope™ Technology.
RNAscope: A Novel In Situ RNA Analysis Platform for Formalin-Fixed Paraffin-Embedded Tissues.

Wang F,Flanagan J, Su N, Wang LC, Bui S, Nielson A, Wu X, Vo HT, Ma XJ, Luo Y (2012). J of Mol Diagnostics, 14(1):22-29.
PMID: 22166544. doi:10.1016/j.jmoldx.2011.08.002.

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