RNAscope® Fluorescent Multiplex Assay

Detecting Drd1a and Drd2 major and minor populations using RNAscope HiPlex assay on same sagittal section of mouse brain

Detecting Drd1a and Drd2 major and minor populations using RNAscope HiPlex assay on same sagittal section of mouse brain

Visualizing striatal Drd1a and Drd2 Medium Spiny Neurons (MSNs) using the RNAscope HiPlex assay for 12 targets in fresh frozen mouse brain sections targets are Drd1a, Drd2, Foxp1, Pcdh8, Synpr (in white), Htr7, Meis2, Calb1, Crym, Cnr1, Wfs1, Th (in yellow)

Visualizing striatal Drd1a and Drd2 Medium Spiny Neurons (MSNs) using the RNAscope HiPlex assay for 12 targets in fresh frozen mouse brain sections targets are Drd1a, Drd2, Foxp1, Pcdh8, Synpr (in white), Htr7, Meis2, Calb1, Crym, Cnr1, Wfs1, Th (in yellow)

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
X
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