RNAscope® Multiplex Fluorescent Assay Options

The RNAscope® Multiplex Fluorescent assays provide the same exceptional sensitivity as our singleplex assays, allowing single-molecule detection of up to four RNA targets simultaneously. The RNAscope® Multiplex Fluorescent assays are ideal for co-localization studies of any genes in nearly any tissue-type using fluorescent labels. Advanced Cell Diagnostics offers two types of multiplex fluorescent assays. RNAscope® Fluorescent Assay, our first generation assay, is an all in one kit, ideal for fresh or fixed frozen tissues. RNAscope® Multiplex Fluorescent Assay v2 is a TSA-based assay ideal for FFPE tissues and requires Akoya Biosciences’ proprietary Opal™ Reagents utilizing Tyramide Signal Amplification (TSA®) technology for visualization.

Sample Images
Four-plex detection of biomarker and checkpoint markers in FFPE in cervical cancer tissues stained with RNAscope<sup>®</sup> Multiplex Fluorescent Assay v2 and RNAscope<sup>®</sup> 4-Plex Ancillary Kit: KRT19 (green), CD8A (yellow), CD4 (red), CD68 (white).

Four-plex detection of biomarker and checkpoint markers in FFPE in cervical cancer tissues stained with RNAscope® Multiplex Fluorescent Assay v2 and RNAscope® 4-Plex Ancillary Kit: KRT19 (green), CD8A (yellow), CD4 (red), CD68 (white).

Four-plex detection of biomarker and checkpoint markers in FFPE lung cancer tissues stained with RNAscope<sup>®</sup> Multiplex Fluorescent Assay v2 and RNAscope<sup>®</sup> 4-Plex Ancillary Kit: KRT19 (green), CD8A (yellow), CD4 (red), CD68 (white).

Four-plex detection of biomarker and checkpoint markers in FFPE lung cancer tissues stained with RNAscope® Multiplex Fluorescent Assay v2 and RNAscope® 4-Plex Ancillary Kit: KRT19 (green), CD8A (yellow), CD4 (red), CD68 (white).

Detection of glutaminergic neurons Vglut1, Vglut2 and GABAergic neurons Vgat expression in the FFPE mouse brain with RNAscope<sup>®</sup> Multiplex Fluorescent Assay v2

Fig. 13: Three-plex detection of glutaminergic neurons, Vglut1 (Red), Vglut2 (Green), and GABAergic neurons Vgat (white) expression in the FFPE mouse brain with RNAscope® Multiplex Fluorescent Assay v2. Nuclei were labeled with DAPI (Blue).

Expresion of Gli-1, Apoe and Lgr5 in mouse cerebellum fresh frozen tissue, RNA in situ hybridization (ISH) using RNAscope<sup>®</sup> Multiplex Fluorescent Assay

Expresion of Gli-1, Apoe and Lgr5 in mouse cerebellum fresh frozen tissue, RNA in situ hybridization (ISH) using RNAscope® Multiplex Fluorescent Assay

Expresion of dopaminergic marker Sncs, Th and Gad1 in mouse substantia nigra fresh frozen tissue, RNA in situ hybridization (ISH) using RNAscope<sup>®</sup> Multiplex Fluorescent Assay

Expresion of dopaminergic marker Sncs, Th and Gad1 in mouse substantia nigra fresh frozen tissue, RNA in situ hybridization (ISH) using RNAscope® Multiplex Fluorescent Assay

Expression of Omp(r) andOlfr73(g) in mouse brain fresh-frozen tissue, RNA in situ hybridization (ISH) using RNAscope<sup>®</sup> Multiplex Fluorescent Assay

Expression of Omp(r) andOlfr73(g) in mouse brain fresh-frozen tissue, RNA in situ hybridization (ISH) using RNAscope® Multiplex Fluorescent Assay

panCK, uPA and Pai-1 mRNA expression in FFPE breast cancer tissue with RNAscope<sup>®</sup> Multiplex Fluorescent Assay

panCK, uPA and Pai-1 mRNA expression in FFPE breast cancer tissue with RNAscope® Multiplex Fluorescent Assay

Fezf2, NPY, and APOE mRNA expression in mouse brain fresh frozen tissue with RNAscope<sup>®</sup> Multiplex Fluorescent Assay

Fezf2, NPY, and APOE mRNA expression in mouse brain fresh frozen tissue with RNAscope® Multiplex Fluorescent Assay

HCV and 18s mRNA expression in HCV infected cells with RNAscope<sup>®</sup> Multiplex Fluorescent Assay

HCV and 18s mRNA expression in HCV infected cells with RNAscope® Multiplex Fluorescent Assay

SNCA and TH63 mRNA expression in mouse cerebellum with RNAscope<sup>®</sup> Multiplex Fluorescent Assay

SNCA and TH63 mRNA expression in mouse cerebellum with RNAscope® Multiplex Fluorescent Assay

Npy and Fexf2 mRNA expression in mouse brain fresh frozen tissue with RNAscope<sup>®</sup> Multiplex Fluorescent Assay

Npy and Fexf2 mRNA expression in mouse brain fresh frozen tissue with RNAscope® Multiplex Fluorescent Assay

IL-8 and beta-Actin mRNA expression in stimulated cultures cells with RNAscope<sup>®</sup> Multiplex Fluorescent Assay

IL-8 and beta-Actin mRNA expression in stimulated cultures cells with RNAscope® Multiplex Fluorescent Assay

Which RNAscope® Fluorescent kit is best for me - two kits are available:

 RNAscope® Multiplex Fluorescent v2RNAscope® Fluorescent Multiplex
Cat no323100320850
DescriptionTSA-based Fluorescent RNAscope® detection assay for up to 4-plex capability using Opal fluorophores (sold separately). Ideal for FFPE tissuesRNAscope® Fluorescent Multiplex Reagent Kit designed as a fluorescent dye for single plex, duplex and up to 3-plex capability. Alexa-488, Atto-550 and Atto-647.
Additional items requiredOpal dyes must be purchased from Akoya Biosciences.None
Fluorophores

Purchased from Akoya Biosciences:

3-plex: 
Opal™ 520 Reagent Pack (PN: FP1487001KT) 
Opal™ 570 Reagent Pack (PN: FP1488001KT)

Opal™ 690 Reagent Pack (PN: FP1497001KT)

4-plex:

Opal™ 520 Reagent Pack (PN: FP1487001KT) 

Opal™ 570 Reagent Pack (PN: FP1488001KT)

Opal™ 620 Reagent Pack (PN: FP1495001KT)

Opal™ 690 Reagent Pack (PN: FP1497001KT)

FITC, Cy3 and Cy5 fluorophores contained within the detection kit (Amp 4)
4-plex enabled4-plex capability enabled with RNAscope® 4-Plex Ancillary Kit for Multiplex Fluorescent v2 (Cat No. 323120)*Available as custom order
Area of researchImmuno-oncology (CD8, PD1, PDL1), Oncology, NeuroscienceNeuroscience
ISH-IF compatibilityCompatible: please refer to Doc# 323100-TNNot recommended
Sample preparation typeFFPE, fixed frozen (low expressors)**
Cells and fresh frozen (if highly autofluorescent)
Fresh frozen, Cells,
Fixed frozen (high expressors)
Gene expression levelLow expressionHigh expression
Auto fluorescenceHigh autofluorescing tissuesLow autofluorescing tissues
Worflow length14 hrs (can split over 2 days)6-8 hours
ProbesRNAscope® Probes: C1, C2, C3, C4RNAscope® Probes: C1, C2, C3
Mounting mediumProlong GoldProlong Gold
Fluorescent microscope with filter set:

Ex Max 358 nm/Em Max 461 nm (DAPI)

Ex Max 494 nm/Em Max 517 nm (FITC)

Ex Max 550 nm/Em Max 570 nm (Cy3)

Ex Max 648 nm/Em Max 667 nm (Cy5)

Ex 358 nm/Em 461 nm (DAPI)

Ex 501 nm/Em 523 nm (FITC)

Ex 554 nm/Em 576 nm (Cy3)

Ex 644 nm/Em 669 nm (Cy5)

*4-plex requires multi-spectral imaging system.

** RNAscope® Multiplex Fluorescent Kit v2 is recommended for high autofluorescing fresh/fixed frozen tissues. Please contact technical support for troubleshooting.

RNAscope® Fluorescent Multiplex Assay(PN 320850)

Technology Overview

RNAscope® Fluorescent Multiplex assay allows simultaneous detection of 3 RNA targets within a single sample using 3 target probes, each optimized for a different probe channel—C1, C2 or C3.  Components within each multiplex probe are unique. 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. Signal detection is performed using dyes with excitation and emission properties equivalent to those of FITC, Cy3 and Cy5 dyes.

Getting Started with Multiplex Assay

The RNAscope® Multiplex Fluorescent Reagent kit contains the Detection Kit, Wash Buffer and the Pretreatment Reagents for fresh frozen tissues.

The RNAscope® Multiplex Fluorescent Manual Assay Workflow Requires:

  • RNAscope® Target Probes (Catalog or Made-to-Order C1, C2 & C3 Probes)
  • RNAscope® Control Probes (Select species-specific positive control C1, C2 & C3 probes and negative control probes)
  • RNAscope® Control Slides (optional)
  • RNAscope® Multiplex Fluorescent Reagent Kit (Pretreatment Kit, Detection kit, and Wash Buffer included)
  • RNAscope® Probe Diluent (Cat. No. 300041) can be used to dilute 50x oligos to 1x for target hybridization of C2 or C3 probes
  • Multispectral fluorescent imaging system (e.g. Nuance FX Multiplex Biomarker Imaging System)
  • HybEZ™ II Hybridization System

Channel selection for multiplexing: three options are available.

Multiplex Fluorescence Color ModuleC1C2C3
Amp4 A ltA
(see example above)
GreenOrangeFar Red
Amp4 A lt BOrangeGreenFar Red
Amp4 A lt COrangeFar RedGreen

Note:  Green: FITC Equivalent, Orange: Cy 3 Equivalent, Far Red: Cy 5 Equivalent

Guidelines for Channel Selection

For the fluorescent multiplex assay, upto 3 target probes could be run in parallel, each designed in a separate probe channel C1 C2 and C3, which are visible in the filters below: 

ChannelProsConsRecommendations
GreenVisible with naked eyeLeast distinct from most tissue autofluorescence*High Expressor
OrangeVisible with naked eyeNone

Low  Expressor or

Expression Level Unknown

Far RedCan be differentiated from autofluorescenceNot visible to the naked eyeLow Expressor

RNAscope® Multiplex Fluorescent Assay v2(PN 323100)

Introducing this new TSA-based assay that enables single-molecule detection of up to four RNA targets simultaneously. The significant signal boost provided by simultaneous background noise suppression from ACD’s patented signal amplification technology combined with Akoya Biosciences TSA® technology allows expanded applications such as dual ISH-IHC and support of FFPE samples.

Technology Overview:

RNAscope® Multiplex Fluorescent Assay v2 allows 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 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. For 4-plex assay we recommend using the Opal 520 (FITC range), Opal 570 (Cy 3 Range), Opal 620 (Texas Red Range) and Opal 690 (Cy 5 Range) dyes. The sequential assay workflow provides flexibility to visualize any probe in any of the TSA linked fluorophore channels.

Getting Started with RNAscope® Multiplex Fluorescent Assay v2

RNAscope® Multiplex Fluorescent Assay v2 to detect up to 3 targets simultaneously requires the following items

  • RNAscope® Multiplex Fluorescent Reagent Kit v2 (Cat. No. 323100). Provides enough reagents to stain ~20 tissue sections, each with an area of approximately 20 mm x 20 mm (0.75in x 0.75in). Each kit contains the RNAscope® Multiplex Fluorescent Detection Kit (Cat. No. 323110), RNAscope H202 and Protease Reagents (Cat. No. 322281), RNAscope Target Retrieval reagents (Cat. No. 322000), RNAscope® Wash Buffer (Cat No. 310091), and RNAscope Multiplex TSA Buffer (Cat. No. 322809).
  • RNAscope® Target Probes in Channel C1, Channel C2, and Channel C3 (C4 Channel probes are required to perform the 4-plex assay).
  • Species specific RNAscope® 3-plex Positive Control Probes and RNAscope 3-plex negative control probe.
  • HybEZ™ Hybridization System with ACD EZ-Batch Slide System (Cat. Nos. 321461 and 321462). Required for RNAscope® hybridization to maintain optimum humidity and temperature at 40ºC.
  • RNAscope® Control Slides (Cat. No. 310045 for Human Cell Pellet; Cat. No. 310023 for Mouse 3T3 Cell Pellet).
  • ImmEdge™ Hydrophobic Barrier Pen (Cat. No. 310018) is the only pen that will maintain a hydrophobic barrier throughout the RNAscope® procedure. No other pen should be used.
  • Additional Reagents/Materials required from ACD for the RNAscope® 4-plex assay
  • RNAscope® 4-Plex Ancillary Kit for Multiplex Fluorescent Kit v2 (Cat No. 323120). Designed to work with the RNAscope® Multiplex Fluorescent Reagent Kit v2 and C1, C2, C3, and C4 Channel Target Probes for 4 plex fluorescent in situ hybridization. It contains HRP-C4 and HRP blocker.
  • Species specific RNAscope® 4-plex Positive Control Probes for RNAscope® Multiplex Fluorescent Assay and RNAscope® 4-plex Multiplex Negative Control Probe Probe (Cat No. 321831).
  • The following Opal™ Dyes can be used; recommended reagent dilution range 1:750-1:3000:
    • Opal 520 Reagent Pack (Part No. FP1487001KT, Akoya Biosciences)
    • Opal 570 Reagent Pack (Part No. FP1488001KT, Akoya Biosciences)
    • Opal 620 Reagent Pack ( Part No. FP1495001KT, Akoya Biosciences)
    • Opal 690 Reagent Pack (Part No. FP1497001KTP, Akoya Biosciences)

Imaging Recommendations

Following are our imaging recommendations for the RNAscope Multiplex v2 assays



3-plex Multiplex FL v2   - multiplexed biomarker imaging system NOT required

Fluorophore

Filter

Opal™ 520

FITC/GFP

Opal™ 570

Cy3

Opal™ 690

Cy5

4-plex Multiplex FL v2 - multiplexed biomarker imaging system

REQUIRED (Nuance FX, Mantra, Vectra 3.0 from Akoya Biosciences)

Fluorophore

Filter

Opal 520

FITC/GFP

Opal 570

Cy3

Opal 620

TexaRed

Opal 690

Cy5

X
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