RNAscope ISH Manual Assays

The manual RNAscope ISH Assay uses a novel and proprietary method of in situ hybridization (ISH) to visualize and spatially detect single RNA molecules at the single cell level, whilst maintaining the spatial tissue microenvironment. The RNAscope ISH manual assays consist of 3 chromogenic assays and 3 fluorescent assays. 

RNAscope 2.5 HD Assay - BROWN

Most sensitive RNA ISH method, used for low expressing gene targets.

Robust, reliable assay, ideal for first. time users.

Intense DAB staining of low copy targets (1-20 copies per cell).

Visible under standard bright-field microscopy.

RNAscope 2.5 HD Assay - RED

Fast Red offers high contrast staining, where DAB is not optimal.

Red dots are clearly distinguished against hematoxylin staining.

Visualized using standard bright-field microscopy.com

RNAscope 2.5 HD Duplex Assay

Enables simultaneous detect of two RNA targets at single-cell resolution.

Utilizes HRP-based GREEN and AP-based Fast RED chromogens.

Permanent staining, ideal for archiving and visible under bright-field microscopy.

Chromogenic RNAscope ISH Technology Assay Table: 

Use the table below to learn more about which manual chromogenic RNA ISH product is right to your research needs. 

*Formalin-fixed, paraffin-embedded (FFPE), Tissue-Microarray (TMA)

 

RNAscope 2.5 HD Assay-BROWN  

RNAscope 2.5 HD Assay-RED  

RNAscope 2.5 HD Duplex Assay  

RNAscope Multiplex Fluorescent Assay  

Two kits are available - please see details here 

Assay Type 

Chromogenic 

Chromogenic 

Chromogenic 

Fluorescent 

Dye Used 

Diaminobenzidine (DAB) 

Fast Red 

HRP Green & Fast Red 

TSA-based Opal fluorophores 

RNAscope Probes Channel Designation 

Channel 1 
(C1 Probes) 

Channel 1 
(C1 Probes) 

Channel 1 & 2 
(C1 & C2 Probes) 

Channel 1-4 
(C1, C2, C3 & C4 Probes) 

Multiplexing 

Singleplex 

Singleplex 

Singleplex, Duplex 

Single to Fourplex 

Key Benefit 

Robust, sensitive, permanent stain. Most widely used RNAscope assay 

Provides bright color stains with high contrast to background 

Utilizes 2 different staining enzymes therefore avoiding any cross talk between the two stains 

Utilizes up to 4 different spectral channels providing high flexibility 

Ideal for 

First time user Routine applications 

Studies of tissues with endogenous color background such as melanin in skin, liver, or lungs from smokers 

Studies interrogating two RNA biomarkers simultaneously 

Co-localization studies of up to 4 genes simultaneously Experimental application requiring flexibility 

Novel Gene or Unknown Expression 

Compatible 

Compatible 

Compatible 

Compatible 

Archival Specimens 

Compatible 

Compatible 

Compatible 

Compatible 

Microscope Imaging System 

Standard bright field 

Standard bright field Multispectral fluorescent imaging 

Standard bright field 

Multispectral fluorescent imaging 

Sample Type* 

FFPE Tissues (TMAs) 
Fixed Frozen Tissues 
Fresh Frozen Tissues 
Cultured cells 

FFPE Tissues (TMAs) 
Fixed Frozen Tissues 
Fresh Frozen Tissues 
Cultured cells 

FFPE Tissues (TMAs) 
Fixed Frozen Tissues 
Fresh Frozen Tissues 
Cultured cells 

FFPE Tissues (TMAs) 
Fixed Frozen Tissues 
Fresh Frozen Tissues 
Cultured cells 

Fluorescent RNAscope ISH Assays

RNAscope HiPlex Assays

Simultaneous detection of up to 12 different RNA targets. 

Validate gene signatures with spatial context within the single cell.

Rapid fluorophore cleaving ensures minimal/no effect on RNA or tissue morphology.

Merge fluorophore images using RNAscope HiPlex Registration Software.

RNAscope Multiplex Fluorescent V2 Assay

Simultaneous detection of up to 4 targets.

Patented ISH signal amplification technology, expands applications to FFPE and ISH-IHC workflows.

Flexible visualization using sequential assay workflow with Opal dyes and Tyramide Signal Amplification technology (TSA™, Akoya Biosciences).

Fluorescent RNAscope ISH Technology Assay Table: 

Use the table below to learn more about which manual fluorescent RNA in situ hybridization product is right for your research needs.

 

RNAscope HiPlex 

RNAscope Multiplex Fluorescent v2 

Plexing offered for target detection 

12 Plex capability enabled with RNAscope 12Plex Ancillary Kit

(Cat. No. 324140) * 

4 Plex capability enabled with RNAscope 4-Plex Ancillary Kit for Multiplex Fluorescent v2

(Cat. No. 323120) * 

Compatible sample preparation 

Fresh frozen, Fixed frozen 

FFPE, fixed frozen (low expressors) ** Cells and fresh frozen

(if highly autofluorescent) 

Additional items required 

None 

Opal dyes must be purchased from Akoya Biosciences. 

Workflow length 

9 hr. (Recommended to split over 3 days) 

14 hr. (Recommended to split over 2 days) 

Fluorophores used/recommended 

Alexa Fluor-488, Atto-550, Atto-647N, Alexa Fluor-750 fluorophores provided in the detection kit (Fluoro reagents) 

Purchased from Akoya Biosciences: 
3-plex: 
Opal™ 520 Reagent Pack (Cat No: FP1487001KT) 
Opal™ 570 Reagent Pack (Cat No: FP1488001KT) 
Opal™ 690 Reagent Pack (Cat No: FP1497001KT) 
 
4-plex: 
Opal™ 520 Reagent Pack (Cat No: FP1487001KT) 
Opal™ 570 Reagent Pack (Cat No: FP1488001KT) 
Opal™ 620 Reagent Pack (Cat No: FP1495001KT) 
Opal™ 690 Reagent Pack (Cat No: FP1497001KT) 

Cleavable Fluorophore 

Yes 

No 

ISH-IF compatibility 

Compatible 

Compatible: please refer to Doc# 323100-TN 

Recommended for 

Low autofluorescing tissues 

High autofluorescing tissues 

Probes Designation 

RNAscope HiPlex Probes: T1, T2, T3, T4, ……T12 

RNAscope Probes: C1, C2, C3, C4 

Recommended Filter sets on the Fluorescent microscope: 

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 Max 753 nm/Em Max 777 nm (Cy7) 

Ex Max 358 nm/Em Max 461 nm (DAPI) 
Ex Max 494 nm/Em Max 525 nm (FITC) 
Ex Max 550 nm/Em Max 570 nm (Cy3) 
Ex Max 588nm/Em Max 616 nm (Tx Red) 
Ex Max 676 nm/Em Max 694 nm (Cy5.5) 

Mounting medium 

Prolong Gold 

Prolong Gold 

Cat No 

324100 

323100 

Description 

RNAscope Fluorescent Multiplex Reagent Kit designed using ACD's proprietary amplification strategy. The assay uses Alexa Fluor-488, Atto-550, Atto-647N and Alexa Fluor-750 for detecting up to 12 targets on the same tissue section. 

TSA-based Fluorescent RNAscope detection assay for up to 4-plex capability using Opal fluorophores (sold separately). Ideal for FFPE tissues 

Area of research 

Neuroscience 

Immuno-oncology (CD8, PD1, PDL1), Oncology, Neuroscience