The RNAscope® Multiplex Fluorescent assays provide the same exceptional sensitivity as our singleplex assays, allowing single-molecule detection of up to four RNA targets simultaneously. The RNAscope® Multiplex Fluorescent assays are ideal for co-localization studies of any genes in nearly any tissue-type using fluorescent labels. Advanced Cell Diagnostics offers two types of multiplex fluorescent assays. RNAscope® Fluorescent Assay, our first generation assay, is an all in one kit, ideal for fresh or fixed frozen tissues. RNAscope® Multiplex Fluorescent Assay v2 is a TSA-based assay ideal for FFPE tissues and requires Perkin Elmer's proprietary Tyramide Signal Amplification technology (TSA™) for visualization.

Sample Images




RNAscope® Fluorescent Multiplex Assay(PN 320850)
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 Multiplex Assay
The RNAscope® Multiplex Fluorescent Reagent kit contains the Detection Kit, Wash Buffer and the Pretreatment Reagents for fresh frozen tissues.

The RNAscope® Multiplex Fluorescent Manual Assay Workflow Requires:

RNAscope® Target Probes (Catalog or Made-to-Order C1, C2 & C3 Probes)
RNAscope® Control Probes (Select species-specific positive control C1, C2 & C3 probes and negative control probes)
RNAscope® Control Slides (optional)
RNAscope® Multiplex Fluorescent Reagent Kit (Pretreatment Kit, Detection kit, and Wash Buffer included)
RNAscope® Probe Diluent (Cat. No. 300041) can be used to dilute 50x oligos to 1x for target hybridization of C2 or C3 probes
Multispectral fluorescent imaging system (e.g. Nuance FX Multiplex Biomarker Imaging System)
HybEZ™ II Hybridization System

RNAscope® Multiplex Fluorescent Assay v2(PN 323100)
Introducing this new TSA-based assay that enables single-molecule detection of up to four RNA targets simultaneously. The significant signal boost provided by simultaneous background noise suppression from ACD’s patented signal amplification technology combined with Perkin Elmer’s TSA® technology allows expanded applications such as dual ISH-IHC and support of FFPE samples.

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 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® Multiplex Fluorescent Reagent Kit v2 (Cat. No. 323100). Provides enough reagents to stain ~20 tissue sections, each with an area of approximately 20 mm x 20 mm (0.75in x 0.75in). Each kit contains the RNAscope® Multiplex Fluorescent Detection Kit (Cat. No. 323110), RNAscope H202 and Protease Reagents (Cat. No. 322281), RNAscope Target Retrieval reagents (Cat. No. 322000), RNAscope® Wash Buffer (Cat No. 310091), and RNAscope Multiplex TSA Buffer (Cat. No. 322809).
RNAscope® Target Probes in Channel C1, Channel C2, and Channel C3 (C4 Channel probes are required to perform the 4-plex assay).
Species specific RNAscope® 3-plex Positive Control Probes and RNAscope 3-plex negative control probe.
HybEZ™ Hybridization System with ACD EZ-Batch Slide System (Cat. Nos. 321461 and 321462). Required for RNAscope® hybridization to maintain optimum humidity and temperature at 40ºC.
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) is the only pen that will maintain a hydrophobic barrier throughout the RNAscope® procedure. No other pen should be used.
Additional Reagents/Materials required from ACD for the RNAscope® 4-plex assay
RNAscope® 4-Plex Ancillary Kit for Multiplex Fluorescent Kit v2 (Cat No. 323120). Designed to work with the RNAscope® Multiplex Fluorescent Reagent Kit v2 and C1, C2, C3, and C4 Channel Target Probes for 4 plex fluorescent in situ hybridization. It contains HRP-C4 and HRP blocker.
Species specific RNAscope® 4-plex Positive Control Probes for RNAscope® Multiplex Fluorescent Assay and RNAscope® 4-plex Multiplex Negative Control Probe Probe (Cat No. 321831).
TSA® Plus fluorescein System; recommended dilution range 1:750 - 1:3000 (Part No. NEL741001KT and NEL741B001KT; Perkin Elmer).
TSA Plus Cyanine 3 System; recommended dilution range 1:750 – 1:3000 (Part No. NEL744001KT and NEL744B001KT; Perkin Elmer).
TSA Plus Cyanine 5 System; recommended dilution range 1:750 – 1:3000 (Part No. NEL745001KT and NEL745B001KT; Perkin Elmer).
The following Opal™ Dyes can be used; recommended reagent dilution range 1:750-1:3000:
Opal 520 Reagent Pack (Part No. FP1487001KT)
Opal 570 Reagent Pack (Part No. FP1488001KT)
Opal 620 Reagent Pack ( Part No. FP1495001KT)
Opal 690 Reagent Pack (Part No. FP1497001KTP)

Bio-Techne Corporation (NASDAQ: TECH) is a leading developer and manufacturer of high quality purified proteins:notably cytokines and growth factors, antibodies, immunoassays, as well as biologically active small molecule compounds and ACD's in situ hybridization detection products,which are sold to biomedical researchers and clinical research laboratories; these operations constitute the core Biotechnology Division, headquartered in Minneapolis, Minnesota. The Protein Platforms Division manufactures innovative protein analysis tools under the ProteinSimple brand name that greatly automate western blotting and immunoassay practices. The Diagnostics Division manufactures FDA-regulated controls, calibrators, blood gas and clinical chemistry controls and other reagents for OEM customer and clinical customers. Bio-Techne products are integral components of scientific investigations into biological processes and the nature and progress of specific diseases. They aid in drug discovery efforts and provide the means for accurate clinical tests and diagnoses. With thousands of products in its portfolio, Bio-Techne generated approximately $563 million in net sales in fiscal 2017 and has approximately 1,800 employees worldwide. For more information on Bio-Techne and its brands, please visit www.bio-techne.com.