RNAscope Multiplex Fluorescent V2 Assay

The RNAscope Multiplex Fluorescent v2 assays provides exceptional sensitivity and allows single-molecule detection of up to four RNA targets simultaneously. The significant signal boost provided by simultaneous background noise suppression from patented signal amplification technology for ISH when combined with Akoya Biosciences proprietary Tyramide Signal Amplification technology (TSA™) for visualization allows expanded applications such as ISH on FFPE tissues and dual ISH-IHC.

Technology Overview:

RNAscope Multiplex Fluorescent Assay v2 allows simultaneous detection of up to four RNA targets within a single sample using up to four target probes and the nuclei can be counter stained and visualized using DAPI fluorescent dye. The four target probes, each designed a different probe channel—C1, C2, C3, or C4 contain several components. 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. For 4-plex assay we recommend using the Opal 520 (FITC range), Opal 570 (Cy 3 Range), Opal 620 (Texas Red Range) and Opal 690 (Cy 5.5 Range) dyes. The sequential assay workflow provides flexibility to visualize any probe in any of the TSA linked fluorophore channels.

Getting Started with RNAscope Multiplex Fluorescent Assay v2

RNAscope Multiplex Fluorescent Assay v2 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 Multiplex Fluorescent Reagent Kit v2 (Cat. No.  323100) (includes Pretreatment Kit, detection kit, wash buffer and TSA dilution buffer)
• RNAscope Control Slides (Cat. No.  310045 for Human Cell Pellet; Cat. No.  310023 for Mouse 3T3 Cell Pellet).
• ImmEdge^® Hydrophobic Barrier Pen (Cat. No.  310018)
• HybEZ™ Hybridization System
• Opal™ or TSA^® Dyes from Akoya Biosciences; recommended dilution Range 1:750 - 1:3000 (to be Purchased Separately by end user)
• Fluorescent microscope with detection capability to detect DAPI, Opal™ 520, Opal™ 570, Opal™ 620 and Opal™ 690

Additional Reagents/Materials required from ACD for the RNAscope 4-plex assay (in addition to the items listed above)

• RNAscope Target Probes (Catalog or Made-to-Order C4 Probes,  not compatible with the RNAscope probes with T4 designation).
• RNAscope 4-Plex Ancillary Kit for Multiplex Fluorescent Kit v2 (Cat. No.  323120). 
• Species specific RNAscope 4-plex Positive Control Probes
• RNAscope 4-plex Multiplex Negative Control Probes (Cat. No.  321831)

Guidelines for Fluorophore Assignment for RNAscope Multiplex Fluorescent v2 Assay

The RNAscope Multiplex Fluorescent v2 Assay is flexible with fluorophore assignments and the end user can choose any fluorophore (from the recommended set) for visualizing any targets, however, fluorophore brightness is an important consideration when assigning them to each target. Please see our recommendations in the table below: