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 Perkin Elmer's proprietary Tyramide Signal Amplification technology (TSA™) 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® Fluorescent MultiplexRNAscope® Multiplex Fluorescent v2
Cat no320850323110
DescriptionRNAscope® 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.TSA-based Fluorescent RNAscope® detection assay for up to 4-plex capability using TSA fluorophores (sold separately). Ideal for FFPE tissues
Additional items requiredNoneTSA® Plus must be purchased from Perkin Elmer
FluorophoresFITC, Cy3 and Cy5 fluorophores contained within the detection kit (Amp 4)

Purchased from Perkin Elmer:
TSA Plus Fluorescein, PN NEL741001KT
TSA Plus Cyanine 3, PN NEL744001KT
TSA Plus Cyanine 5, NEL745001KT

4-plex enabledAvailable as custom order4-plex capability enabled with RNAscope® 4-Plex Ancillary Kit for Multiplex Fluorescent v2 (Cat No. 323120)*
Area of researchNeuroscienceImmuno-oncology (CD8, PD1, PDL1), Oncology, Neuroscience
Sample preparation typeFresh, fixed frozenFFPE **
Gene expression levelHigh expressionLow expression
Auto fluorescenceLow autofluorescing tissuesHigh autofluorescing tissues
Worflow length6-8 hours14 hrs (can split over 2 days)
ProbesRNAscope® Probes: C1, C2, C3RNAscope® Probes: C1, C2, C3, C4
Mounting mediumProlong GoldProlong Gold
Fluorescent microscope with filter set:

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)

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)

*4-plex requires use of opal 6-color fIHC (nel796001kt) or opal 7-color fIHC (nel797001kt, nel797b001kt) kit (which contains: OPAL 520, OPAL 570, OPAL 620, OPAL 690); multi spectral imaging capabilities required.

** RNAscope® Multiplex Fluorescent Kit v2 can also be used on high autofluorescing fixed/fresh frozen tissues and/or targets that are low expressors.

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 (eg Nuance FX Multiplex Biomarker Imaging System)
  • HybEZ™ 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 Perkin Elmer’s 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).
  • TSA® Plus fluorescein System; recommended dilution range 1:750 - 1:3000 (Part No. NEL741001KT and NEL741001KTK; Perkin Elmer).
  • TSA Plus Cyanine 3 System; recommended dilution range 1:750 – 1:3000 (Part No. NEL744001KT and NEL744B001KT; Perkin Elmer).
  • TSA Plus Cyanine 5 System; recommended dilution range 1:750 – 1:3000 (Part No. NEL745001KT and NEL745B001KT; Perkin Elmer).
  • The following Opal™ Dyes can be used; recommended reagent dilution range 1:750-1:3000:
    • Opal 520 Reagent Pack (Part No. FP1487001KT)
    • Opal 570 Reagent Pack (Part No. FP1488001KT)
    • Opal 620 Reagent Pack ( Part No. FP1495001KT)
    • Opal 690 Reagent Pack (Part No. FP1497001KTP)

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

Fluorescein

FITC/GFP

Cyanine 3

Cy3

Cyanine 5

Cy5

4-plex Multiplex FL v2 - multiplexed biomarker imaging system

REQUIRED (Nuance, Mantra, Vectra from PerkinElmer)

Fluorophore

Filter

Opal 520

FITC/GFP

Opal 570

Cy3

Opal 620

TexaRed

Opal 690

Cy5

X
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