RNAscope® Fluorescent Multiplex Assays
Spatial analysis of gene expression is an essential tool for comprehensive studies of complex, highly heterogeneous tissues, such as the brain, tumors, and developing organs. Traditional RNA-ISH can be challenging due to low sensitivity, inconsistent detection accompanied with limited plexing. RNAscope® Fluorescent Multiplex assays provide exceptional sensitivity, allowing single-molecule detection of RNA targets at the single cell level. The RNAscope® Multiplex Fluorescent assays are ideal for simultaneous detection studies of any genes in nearly any tissue-type using fluorescent labels. We offer with various levels of plexing to:
- FIND cells of interest by using cell specific markers
- CONFIRM cells by simultaneous detection of additional targets
- CHARACTERIZE cellular heterogeneity of complex tissues by mapping and identifying multiple cell subtypes simultaneously
“Adding a histological context to scRNAseq data is important for fully understanding the biology of distinct cell populations, and HiPlex RNAscope is a great way to do this. The ability to stain a single section of tissue with up to 12 mRNA probes makes HiPlex RNAscope a powerful tool for distinguishing cells types in situ. While the HiPlex RNAscope protocol is relatively straightforward to conduct, ACD Bio has always been there to help when I need sales or technical assistance. I would recommend HiPlex RNAscope to anyone looking to visualize distinct populations of cells from scRNAseq data in situ.”
-Jocob Swanson, Hospital for special surgery, NY
RNAscope® In Situ Hybridization Assay Workflow
Step 1:
Permeabilize
Tissue sections or cells are fixed onto slides and pretreated with RNAscope® Pretreatment Kit to unmask target RNA and permeabilize cells.
Step 2:
Hybridize
Designed with ~20 target-specific double Z probes, RNAscope® Probes hybridize to target RNA molecules.
Step 3:
Amplify
RNAscope® Detection Reagents amplify the hybridization signals via sequential hybridization of amplifiers and label probes.
Step 4:
Visualize
Each punctate dot signal represents a single test target RNA molecule and can be visualized with a microscope.
Step 5:
Quantify
Single-molecule signals can be quantified on a cell-by-cell basis by manual counting or automated image analysis using HALO Software.
Which RNAscope® Multiplex kit is best for me - three kits are available
Use the table below to learn more about which RNA in situ hybridization product is right for your research needs.
RNAscope® HiPlex | RNAscope® Multiplex Fluorescent v2 | RNAscope® Fluorescent Multiplex | |
|---|---|---|---|
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)* | 3 Plex capability |
Compatible sample preparation | Fresh frozen, Fixed frozen | FFPE, fixed frozen (low expressors)** Cells and fresh frozen (if highly autofluorescent) | Fresh frozen, Cells, Fixed frozen (high expressors) |
Additional items required | None | Opal dyes must be purchased from Akoya Biosciences. | None |
Workflow length | 9 hr (Recommended to split over 3 days) | 14 hr (Recommended to split over 2 days) | 6-8 hours |
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: | Alexa Fluor-488, Atto-550 and Atto-647N fluorophores provided in the detection kit (Amp 4) |
Cleavable Fluorophore | Yes | No | No |
ISH-IF compatibility | Compatible | Compatible: please refer to Doc# 323100-TN | Not recommended |
Recommended for | Low autofluorescing tissues | High autofluorescing tissues | Low autofluorescing tissues |
Probes Designation | RNAscope® HiPlex Probes: T1, T2, T3, T4, ……T12 | RNAscope® Probes: C1, C2, C3, C4 | RNAscope® Probes: C1, C2, C3 |
Recommended Filter sets on the Fluorescent microscope: | Ex Max 358 nm/Em Max 461 nm (DAPI) | Ex Max 358 nm/Em Max 461 nm (DAPI) | Ex Max 358 nm/Em Max 461 nm (DAPI) |
Mounting medium | Prolong Gold | Prolong Gold | Prolong Gold |
Cat No | 324100 | 323100 | 320850 |
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 | RNAscope® Fluorescent Multiplex Reagent Kit employs Alexa Fluor-488, Atto-550 and Atto-647N fluorophores for up to 3-plex detection capability. |
Area of research | Neuroscience | Immuno-oncology (CD8, PD1, PDL1), Oncology, Neuroscience | Neuroscience |
