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RNAscope® Fluorescent Multiplex Assay

Detection of Drd1a and DRD2 mRNA using RNAscope<sup>®</sup> Fluorescent Multiplex assay on fresh frozen tissue samples from normal mouse brain striatum

Detection of Drd1a and DRD2 mRNA using RNAscope® Fluorescent Multiplex assay on fresh frozen tissue samples from normal mouse brain striatum

Detection of G protein-coupled receptors mRNA using RNAscope<sup>®</sup> Fluorescent Multiplex assay on fresh frozen tissue samples from normal mouse brain hippocampus

Detection of G protein-coupled receptors mRNA using RNAscope® Fluorescent Multiplex assay on fresh frozen tissue samples from normal mouse brain hippocampus

RNAscope® Fluorescent Multiplex Assay is our first multiplex assay and is based on our patented signal amplification and background suppression technology. The assay offers a novel and proprietary method of in situ hybridization (ISH) to achieve single cell resolution for RNA detection in a multitude of slide mounted samples.

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 RNAscope® Fluorescent Multiplex Assay

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

  • RNAscope® Target Probes (Catalog or Made-to-Order C1 to C3 Probes,  not compatible with the RNAscope® probes with T1-T3 designation).
  • Species specific RNAscope® 3-plex Positive Control Probes
  • RNAscope® 3-plex negative control probes (Cat. No.  320871)
  • RNAscope® Probe Diluent (Cat. No.  300041)
  • RNAscope® Fluorescent Multiplex Reagent Kit (Cat. No.  320850) (includes Pretreatment Kit, detection kit, and wash buffer)
  • Immedge® Hydrophobic Barrier Pen (Cat. No.  310018)
  • HybEZ™ Hybridization System
  • Fluorescent microscope with detection capability to detect DAPI, AF488, Atto550, and Atto647N

Channel selection for multiplexing: three modules are available.

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

 

Guidelines for Fluorophore Assignment for RNAscope® Fluorescent Multiplex Assay

The RNAscope® Fluorescent Multiplex Assay offers three Alternate display modules and the end user can choose Alt A, B or C for visualizing the targets, however, fluorophore brightness is an important consideration when assigning them to each target. Please see our recommendations in the table below:

Microscopy ChannelProsConsRecommendations
Fluorescein - Green
For Alexa Fluor 488
Visible with naked eyeLeast distinct from most tissue autofluorescenceHigh Expressor
Cyanine 3 - Orange
For Atto 550
Visible with naked eyeNoneLow Expressor or Expression Level Unknown
Cyanine 5 - Far Red
For Atto 647N
Easily differentiated from autofluorescenceNot visible to the naked eyeLow Expressor
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