Manual assays


Four flexible solutions for all your research needs

Independent of your genes of interest, our easy RNA in situ hybridization assay uses common reagents and protocols; providing universal assay conditions. All RNA-ISH kits contain reagents in a convenient ready-to-use (RTU) format, and chromogenic dyes are available to interrogate a single or two genes simultaneously. Most versatile are our RNAscope® Multiplex Fluorescent Reagent Kits, enabling you to interrogate 1, 2 or 3 genes simultaneously.


Automated RNAscope® ISH assays are also available for Leica Systems (LS) and Ventana Systems (VS).


Which RNAscope® Assay is right for me?


 

RNAscope® 2.5 HD Assay-BROWN 

RNAscope® 2.5 HD Assay-RED 

RNAscope® 2.5 HD Duplex Assay 

RNAscope® Multiplex Fluorescent Assay 

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 upto 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 upto 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

RNAscope 2.5 HD Assay - BROWN


The RNAscope® 2.5 High Definition (HD)—BROWN Assay, is based on ACD’s patented signal amplification and background suppression technology. The assay uses a novel and proprietary method of in situ hybridization (ISH) to visualize single RNA molecules per cell in a multitude of sample types mounted on slides. RNAscope® Assays do not require the RNA-free environment used for traditional ISH. The HD version is the most sensitive RNA ISH method and can be used for low expressing gene targets.


RNAscope® 2.5 HD Reagent Kit—BROWN is the ideal starter kit for first time users. This very robust assay gives reliable results which are ideal for archiving due to the permanent staining. It is the standard in molecular pathology diagnostics, usable for a wide source of samples and the results are visible under any standard bright field microscope. The RNAscope® 2.5 HD - Brown Reagent Kits are designed to provide more intense Diaminobenzidine (DAB) staining when low copy target gene expression is anticipated (1–20 copies per cell). The RNAscope® 2.5 HD reagent kits are compatible with the same RNAscope® probes used previously with RNAscope® 2.5 assays.

The RNAscope® 2.5 HD—BROWN Manual Assay Workflow Requires:


  1. RNAscope® Target Probes (Catalog or Made-to-Order channel 1 (C1) Probes)
  2. RNAscope® Control Probes (Select species-specific positive control probes and negative control probes)
  3. RNAscope® Control Slides
  4. RNAscope® 2.5 HD Reagent Kit—BROWN (Pretreatment Kit, Detection kit, and Wash Buffer included)
  5. Brightfield microscope
  6. HybEZ™ Hybridization System

RNAscope 2.5 HD Assay - RED


The RNAscope® 2.5 High Definition (HD)—Red Assay, is based on ACD’s patented signal amplification and background suppression technology. The assay uses a novel and proprietary method of in situ hybridization (ISH) to visualize single RNA molecules per cell in a multitude of sample types mounted on slides. RNAscope® Assays do not require the RNA-free environment used for traditional ISH. The HD version is the most sensitive RNA ISH method and can be used for low expressing gene targets.


The RNAscope® 2.5 HD Reagent Kit-RED assay with it Fast Red dye offers a higher contrast and is the first choice in situ hybridization applications where chromogenic staining with DAB is less desirable, such as staining of highly pigmented lung, liver, retina and melanoma tissue specimens. Also for genes where a lower expression is assumed ACD recommends this assay as the red dots clearly distinguish against the hematoxylin staining and are visible under are standard brightfield microscope.

The RNAscope® 2.5 HD--RED Manual Assay Workflow Requires:


  1. RNAscope® Target Probes (Catalog or Made-to-Order C1 Probes)
  2. RNAscope® Control Probes (Select species-specific positive control probes and negative control probes)
  3. RNAscope® Control Slides (optional)
  4. RNAscope® 2.5 HD Reagent Kit—RED (Pretreatment Kit, Detection kit, and Wash Buffer included)
  5. Brightfield microscope or multispectral fluorescent imaging system
  6. HybEZ™ Hybridization System

RNAscope® 2.5 HD Duplex Assay


The RNAscope® 2.5 HD Duplex Assay is based on ACD’s patented signal amplification and background suppression technology. The assay uses a novel and proprietary method of in situ hybridization (ISH) to visualize single RNA molecules per cell in FFPE samples mounted on slides. RNAscope® Assays do not require the RNA-free environment used for traditional ISH. RNAscope® probes in Channel 1 and Channel 2 are compatible with this assay which utilizes HRP-based Green and AP-based Fast Red chromogens to result in a detectable green and red signal respectively. The RNAscope® 2.5 HD Duplex Assay can be used for simultaneous visualization of two RNA targets while maintaining single cell resolution. The applications of this assay include study of gene co-regulation, profile gene expression in a specific cell type (identified using a cell specific marker) and map coexpression of two targets within the same cellular context based on reliable chromogenic detection of the two transcripts.


RNAscope® 2.5 HD Duplex Reagent Kit gives reliable results which are ideal for archiving due to the permanent staining visible under any standard bright field microscope. The RNAscope® 2.5 HD Duplex Reagent Kits are designed to provide more intense staining when low copy target gene expression is anticipated (1–20 copies per cell). The RNAscope® 2.5 HD Duplex reagent kits are compatible with the same RNAscope® probes used previously with RNAscope®2.0 2-plex assays.

The RNAscope® 2.5 HD Duplex Manual Assay Workflow Requires:


  1. RNAscope® Target Probes (Catalog or Made-to-Order C1 & C2 Probes,  compatitle with the same RNAscope® probes used previously with RNAscope®2.0 2-plex assays.)
  2. RNAscope® Duplex Positive Control Probes
  3. RNAscope® Duplex Negative Control Probes (Cat. No. 320751)
  4. RNAscope® Control Slides (optional)
  5. RNAscope® 2.5 HD Duplex Reagent Kit (Cat. no 322430) (Includes Pretreatment Kit for FFPE samples, detection kit and wash buffer)
  6. Vector Labs Vectamount Permanent Mounting Medium (Cat. No. 321584)
  7. ImmEdge Hydrophobic Barrier Pen (Cat. No 310018)
  8. HybEZ™ Hybridization System
  9. Brightfield microscope