RNAScope® Multiplex Fluorescent V2 Assay

Detecting intestinal stem cell population in the base of the crypt using the RNAscope multiplex fluorescent assay. Shown here are known markers of the intestinal stem cells, with Lgr5 in white, Ascl2 in green, and Olfm4 in red.

Detecting intestinal stem cell population in the base of the crypt using the RNAscope multiplex fluorescent assay. Shown here are known markers of the intestinal stem cells, with Lgr5 in white, Ascl2 in green, and Olfm4 in red.

Immune infiltration and state of lymphoid cells in the TME: detecting CD4mRNA in green, FOXP3 mRNA in yellow, IFNg mRNA in red and CD8 antibody in white. The slides are stained using RNAscope<sup>®</sup> Multiplex Fluorescent v2 assay on FFPE tissue samples

Immune infiltration and state of lymphoid cells in the TME: detecting CD4mRNA in green, FOXP3 mRNA in yellow, IFNg mRNA in red and CD8 antibody in white. The slides are stained using RNAscope® Multiplex Fluorescent v2 assay on FFPE tissue samples

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:

Microscopy ChannelProsConsRecommendations
Fluorescein - Green
For Opal™ 520
Visible with naked eyeLeast distinct from most tissue autofluorescenceHigh Expressor
Cyanine 3 - Orange
For Opal™ 570
Visible with naked eyeNoneLow Expressor or Expression Level Unknown
Texas Red - Red
For Opal™ 620
Easily differentiated from autofluorescenceNoneLow Expressor or Expression Level Unknown
Cyanine 5.5 - Near IR
For Opal™ 690
Easily differentiated from autofluorescenceNot visible to the naked eyeLow Expressor
X
Contact Us

Complete one of the two forms below and we will get back to you.

Advanced Cell Diagnostics

Our new headquarters office starting May 2016:

7707 Gateway Blvd.  
Newark, CA 94560
Toll Free: 1 (877) 576-3636
Phone: (510) 576-8800
Fax: (510) 576-8798

 

Bio-Techne

19 Barton Lane  
Abingdon Science Park
Abingdon
OX14 3NB
United Kingdom
Phone 2: +44 1235 529449
Fax: +44 1235 533420

 

 

Advanced Cell Diagnostics China

Building 15, No. 26 Xihuan South Road
Beijing Economic-Technological Development Area
Beijing, China, Zip code: 100176

400-966-1796
info_china.ACD@bio-techne.com
For general information: Leon.Li@bio-techne.com
Web: www.acdbio.com/cn

For general information: Info.ACD@bio-techne.com
For place an order: order.ACD@bio-techne.com
For product support: support.ACD@bio-techne.com
For career opportunities: hr.ACD@bio-techne.com