Product Overview
The RNAscope Multiplex Fluorescent assays provide exceptional sensitivity and allows single-molecule detection of up to four RNA targets simultaneously. The significant signal boost provided by simultaneous background noise suppression from patented signal amplification technology for ISH when combined with Tyramide Signal Amplification (TSA) technology for visualization allows expanded applications such as ISH on FFPE tissues and dual ISH-IHC.
The new Pretreat Pro reagent now enabled on RNAscope Multiplex manual assay allows for a protease-free solution to successfully detect target proteins without compromising on signal quality.
Protease-Free Workflow
Protease-Free Workflow
- Expanded protein codetection
- Preserved tissue morphology
- Flexible assay design
- Optimized for both Frozen and FFPE samples
Fluorescent Multiplex Detection
Fluorescent Multiplex Detection
- Visualize up to 4 targets
- Multiple fluorophore options
Flexible Assay Design
Flexible Assay Design
- Automated or manual workflows available
- Choose any 3 RNAscope probes
- Enables addition of 4th target
- Open fluorophore assignment
Technology Overview
RNAscope Multiplex Fluorescent assays allow simultaneous detection of up to four RNA targets within a single sample using up to four target probes and the nuclei can be counter stained and visualized using DAPI fluorescent dye. The four target probes, each designed for a different probe channel - C1, C2, C3, or C4 - contain several components. The lower part of the double Z probe is designed to hybridize to a specific RNA target. The upper part of the double Z probe, along with the preamplifiers, amplifiers and dyes are assigned to a specific probe channel. Different fluorophores are assigned to the different channels depending on the TSA Vivid™, Opal™ or TSA plus fluorophore selected for that channel. The sequential assay workflow provides flexibility to visualize any probe in any of the TSA linked fluorophore channels.
Featured Applications
Immunology/Inflammation
Immunology/Inflammation
- Identification of cytokines and their cellular origin
- Detection of long non-coding RNA (lncRNA) in inflammatory diseases
- Therapeutic potential of secreted proteins in inflammatory diseases
Neuroscience
Neuroscience
- Detect, characterize, and (co-) localize neuronal cell types
- Detect G protein-coupled receptors (GPCRs)
RNA Biomarkers
RNA Biomarkers
- Develop species-specific probes for known cancer biomarkers
- Quickly verify expression of RNA biomarkers
Our Automated Assay Designed for Leica BOND RX is Multiomic!
Same RNA chemistry
Enhanced capability
Check out our RNAscope LS Multiomic Assay
To proceed with RNAscope™ LS Multiplex Fluorescent Assay, view ordering information. | Prefer to work at your bench? View ordering information for the manual assay. |
Images using the RNAscope Multiplex v2 assay
Customer Success Stories
University of Pittsburg
Max Planck Florida Institute for Neuroscience
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Resources
Watch our Webinar: Spatial Expert Insights: Decoding Host Microbial Interactions That Influence Barrier Tissue Immunity.
The RNAscope LS Multiplex Fluorescent Assay and RNAscope Multiplex Fluorescent v2 supports interrogation of up to 4 target probes, each designed in a separate probe channel C1, C2, C3 and C4, which are visible in the filters associated with the dyes such as FITC, Cy3, Cy5 and Texas Red. For viewing of 4 plex assay, a multiplexed biomarker imaging system is required, such as Phenoimgaer from Akoya Biosciences.
Target fluorophore assignments are flexible, so probe channel does not need to be selected based on expression level. Fluorophore brightness is an important consideration when assigning them to each target. Please see our recommendations in the table below:
Filter | Dye | 3-plex options | 4-plex options | Visible Spectrum | Expression recommendation | Notes |
| FITC | TSA Vivid 520 Opal 520 | √
| √
| √ | High | Least distinct from autofluorescence |
| Cy3 | TSA Vivid 570 Opal 570 | √
| √
| √
| Any | |
| Texas Red | Opal 620 | √
| √
| Any | ||
| Cy5 | TSA Vivid 650 Opal 690 | √
| √
| - | Any (recommended for lowest expressor in panel) | |
| Cy7 | Opal 780 | √
| √
| - | Medium to high | •Must be developed last •Requires near IR filter |
RNAscope™ LS Multiplex Fluorescent Assay | RNAscope™ Multiplex Fluorescent V2
RNAscope™ LS Multiplex Fluorescent Assay
ACD Components | # of Slides | Catalog # |
RNAscope™ LS Multiplex Fluorescent Reagent Kit with TSA Vivid Dyes •Pretreatment & Detection Reagents •RNAscope LS Multiplex TSA Buffer Pack •TSA Vivid Fluorophore 520 (PN 323271), TSA Vivid Fluorophore 570 (PN 323272) and TSA Vivid Fluorophore 650 (PN 323273). | 60 | 323275 |
RNAscope LS Multiplex Reagent Kit •Pretreatment & Detection Reagents •RNAscope LS Multiplex TSA Buffer Pack
| 60 | 322800 |
RNAscope LS 4-Plex Ancillary Kit Multiplex Reagent Kit (cannot be used alone) •Designed to work with the RNAscope LS Multiplex Fluorescent Reagent Kit • contains HRP-C4 and HRP blocker | 60 | 322830 |
| RNAscope LS 2.5 Target Probes (Catalog or Made-to-Order C2, C3 & C4 LS 2.5 Probes) | 30 | Various |
| RNAscope 3-plex LS Multiplex Control Positive Probe- Hs POLR2A (C1), PPIB (C2), UBC(C3) | 30 | 320868 |
| RNAscope 3-plex LS Multiplex Control Positive Probe - Mm polr2A (C1), ppib(C2), ubc(C3) | 30 | 320888 |
| RNAscope 4-plex LS Multiplex Control Positive Probe -Hs | 30 | 321808 |
| RNAscope 4-plex LS Multiplex Control Positive Probe -Mm | 30 | 321818 |
| RNAscope 4-plex LS Multiplex Control Positive Probe -Rn | 30 | 321828 |
| RNAscope 3-plex LS Multiplex Negative Control | 30 | 320878 |
| TSA Vivid 520nm | See Channel Selection guide for options>> | 323271 |
| TSA Vivid 570nm | 323272 | |
| TSA Vivid 650nm | 323273 | |
Consumables from Akoya Biosciences | Catalog # | |
| Opal 520 Reagent Pack | See Channel Selection guide for options>>
| FP1487001KT |
| Opal 570 Reagent Pack | FP1488001KT | |
| Opal 620 Reagent Pack | FP1495001KT | |
| Opal 650 Reagent Pack | FP1496001KT | |
| Opal 690 Reagent Pack | FP1497001KT | |
Opal Polaris 780 Reagent Pack Antibody Diluent/Block (if Opal Polaris 780 is used)
| FP1501001KT ARD1001EA | |
Consumables from Leica Biosystems | Catalog # | |
| BOND 30 mL Open containers | OP309700 | |
| BOND 6 mL Titration kit | OPT9049 | |
| BOND Research Detection System (2) | DS9455/DS9777 | |
| BOND Universal Covertile | S21.4611/S21.2001 | |
| BOND Epitope Retrieval Solution 1-1L (RTU) | AR9961 | |
| BOND Epitope Retrieval Solution 2-1L (RTU) | AR9640 | |
| BOND Dewax Solution – 1L (RTU) | AR9222 | |
| BOND Wash Solution 10X Concentrate – 1L | AR9590 | |
| BOND Aspirating Probe Cleaning System | CS9100 | |
| BOND Mixing Stations |
S21.1971 | |
RNAscope™ Multiplex Fluorescent V2
ACD Components | # of Slides | Catalog # |
RNAscope Multiplex Fluorescent Reagent Kit v2 •Pretreatment, Wash & Detection Reagents •RNAscope Multiplex TSA Buffer
| 20 | 323100 |
RNAscope 4-Plex Ancillary Kit Multiplex Reagent Kit (cannot be used alone) •Designed to work with the RNAscope Multiplex Fluorescent Reagent Kit • contains HRP-C4 and HRP blocker | 20 | 323120 |
| RNAscope 2.5 Target Probes (Catalog or Made-to-Order C2, C3 & C4 LS 2.5 Probes) | 20 | Various |
| RNAscope 3-plex LS Multiplex Control Positive Probe- Hs POLR2A (C1), PPIB (C2), UBC(C3) | 320868 | |
| RNAscope 3-plex LS Multiplex Control Positive Probe - Mm polr2A (C1), ppib(C2), ubc(C3) | 320888 | |
| RNAscope 4-plex LS Multiplex Control Positive Probe - Hs | 321808 | |
| RNAscope 4-plex LS Multiplex Control Positive Probe - Mm | 321818 | |
| RNAscope 4-plex LS Multiplex Control Positive Probe -Rn | 321828 | |
| RNAscope 3-plex Multiplex Negative Control | 20 | 320871 |
| TSA Vivid 520nm | See Channel Selection guide for options>> | 323271 |
| TSA Vivid 570nm | 323272 | |
| TSA Vivid 650nm | 323273 | |
| RNAscope Probe Diluent | 300041 | |
| ImmEdge® Hydrophobic Barrier Pen | 310018 | |
Consumables from Akoya Biosciences | Catalog # | |
| Opal 520 Reagent Pack | See Channel Selection guide for options>>
| FP1487001KT |
| Opal 570 Reagent Pack | FP1488001KT | |
| Opal 620 Reagent Pack | FP1495001KT | |
| Opal 650 Reagent Pack | FP1496001KT | |
| Opal 690 Reagent Pack | FP1497001KT | |
Opal Polaris 780 Reagent Pack Antibody Diluent/Block (if Opal Polaris 780 is used)
| FP1501001KT ARD1001EA | |