RNA expression of TNFSF4 (OX40L) (green), CD274 (PDL1) (red) and HAVCR2 (TIM3) (white) using RNAscope LS Multiplex Fluorescent Assay
RNA expression of Malat1 (red, high in nuclear) and Mog (white) in fresh frozen rat brain tissue using RNAscope LS multiplex Fluorescent Assay.
Positive control probes demonstrate RNA expression of Tbp (green), PPib (red) and UBC (white) in mouse brain fresh frozen tissue using RNAscope LS Multiplex Fluorescent Assay
RNA expression of CD3D (green) CD8A (red) combined with protein expression of CDC (white) (IHC) in human tonsil FFPE tissue using RNAscope LS Multiplex Fluorescent assay
RNA expression of Sc17a6-C1 (Vglut2) (green), Slc17a7-C2 (Vlgut1) (red) and Slc32a1-C3 (Vgat) in mouse brain fresh frozen tissue using RNAscope LS Multiplex Fluorescent Assay
RNA expression of Cnr1(green), Drd1 (red) and Nmdar1 (white) in mouse brain FFPE tissue using RNAscope LS Multiplex Fluorescent Assay
RNA expression of Sc17a6 (Vglut2)(green), SLc17a7 (Vlgut1) (red) and Slc32a1 (white) in mouse brain fresh frozen tissue using RNAscope LS Multiplex Fluorescent Assay
RNA expression of HPV HR18(green), Hs-CDKN2A (p16) (red) and Hs-MKI67(white) in human head and neck cancer FFPE tissue using RNAscope LS Multiplex Fluorescent Assay
RNA expression of KRT19(green), PECAM1 (red) and MKI67(white) in human breast cancer FFPE tissue using RNAscope LS Multiplex Fluorescent Assay
RNA expression of CD8A (green) CD274 (red) combined with protein expression of CD8 (white, IHC) in human lung cancer FFPE sample using RNAscope LS Multiplex Fluorescent Assay
RNA expression of TNFSF4 (OX40L)(green), CD274 (PDL1) (red) and HAVCR2 (TIM3) (white)in human lung cancer FFPE tissue using RNAscope LS Multiplex Fluorescent Assay
RNA expression of CXCL13 (green), CD274 (PDL1) (red) and KRT`9-C3 (white) in human lung cancer FFPE tissue using RNAscope LS Multiplex Fluorescent Assay
RNA expression of IDO1 (green), CD274 (PDL1)(red) and CTLA4 (white)in human lung cancer FFPE tissue using RNAscope LS Multiplex Fluorescent Assay
RNA expression of PTPRC (CD45) (green), IFNG (red) and of CD274 (PDL1) (white) in human lung cancer FFPE tissue using RNAscope LS Multiplex Fluorescent Assay
Technology Overview
RNAscope LS Multiplex Fluorescent Assay 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. Signal detection is performed by Tyramide linked Fluorescein, Cy3 and Cy5 dyes. The sequential assay workflow provides flexibility to visualize any probe in any of the TSA linked fluorophore channels. The combination of ACD patented signal amplification system and the tyramide technology provides a significant boost in the signal while suppressing background noise. This feature makes the assay suitable for use on both FFPE and fresh-frozen samples. It is ideal for co-localization applications and compatible with dual ISH IHC protocols.
Guidelines for Microscope Channel Selection
The RNAscope LS Multiplex Fluorescent Assay supports interrogation of up to 4 target probes, each designed in a separate probe channel C1, C2, C3 and C4, which are visible in the filters associated with the dyes such as FITC, Cy3, Cy5 and Texas Red. The signal amplification in all 4 channels is balanced, removing the necessity of selecting channels based on gene-specific expression level. Customers can freely select channels for their genes. For viewing of 4 plex assay, a multiplexed biomarker imaging system is required, such as Nuance FX Multiplex Biomarker Imaging System, Vectra 3.0 Automated Quantitative Pathology Imaging System or Mantra Quantitative Pathology Workstation from Perkin Elmer.
| 3-plex fluorophore | 3-plex filter | 4-plex fluorophore | 4-plex filter |
|---|
TSA Vivid 520/Opal 520 TSA Vivid 570/Opal 570 TSA Vivid 650/Opal 690 | FITC/GFP Cy3 Cy5 | Opal 520 Opal 570 Opal 620 Opal 690 | FITC/GFP Cy3 Texas Red Cy5 |
What to order
Visualization requires a multispectral fluorescent imaging system (e.g. Nuance FX Multiplex Biomarker Imaging System)
Order the following components from ACD:
| Component | Cat. No. | Description |
|---|
| RNAscope™ LS Multiplex Fluorescent Reagent Kit with TSA Vivid Dyes | 323275 | RNAscope LS (Leica Biosystems) Multiplex assay TSA Vivid Dyes is a RNA in situ hybridization (ISH) fluorescent assay for use of three fluorescent dyes for single plex, duplex and up to 3-plex capability with the Leica Biosystems BOND RX automated IHC/ISH slide staining systems (Note: Using Leica Software v BDZ 11). The kit includes the pretreatment, TSA buffer (322810), and detection reagents for automated amplification and TSA Vivid Fluorophore 520 (PN 323271), TSA Vivid Fluorophore 570 (PN 323272) and TSA Vivid Fluorophore 650 (PN 323273). . Each reagent kit provides enough reagents to stain ~60 slides. Leica Research Kit (DS9455) , is also required to run the RNAscope Multiplex Fluorescent Kit. |
RNAscope LS Multiplex Reagent Kit Contains pretreatment reagents, TSA buffer (322810) and detection reagents for ~60 slides This kit is required for 3-plex and 4-plex | 322800 | |
RNAscope LS 4-Plex Ancillary Kit Multiplex Reagent Kit Designed to work with the RNAscope LS Multiplex Fluorescent Reagent Kit (Cat. No. 322800), C1, C2, C3 and C4 Channel Target Probes for 4 plex fluorescent in situ hybridization. It contains HRP-C4 and HRP blocker. | 322830 | |
RNAscope LS 2.5 Target Probes (Catalog or Made-to-Order C1, C2, C3 & C4 LS 2.5 Probes) | | 300038 RNAscope 2.5 LS Probe (RTU) | 300038 | 300038-C2 RNAscope 2.5 LS Probe- C2 (50x) | 300038-C2 | 300038-C3 RNAscope 2.5 LS Probe- C3 (50x) | 300038-C3 | 300038-C4 RNAscope 2.5 LS Probe- C4 (50x) | 300038-C4 |
| |
RNAscope LS 2.5 Positive Control Probes Select species-specific positive control C1, C2 & C3
probes and negative control probes | | |
320868 - RNAscope 3-plex LS Multiplex Control Positive Probe- Hs POLR2A (C1), PPIB (C2), UBC(C3) | 320868 | 320888 - RNAscope 3-plex LS Multiplex Control Positive Probe - Mm polr2A (C1), ppib(C2), ubc(C3) | 320888 | 320838 - RNAscope 3-plex LS Multiplex Control Positive Probe - HeLa Cells TBP (C1), PPIB (C2), POLR2A (C3) | 320838 | RNAscope 4-plex LS Multiplex Control Positive Probe - POLR2A (C1), PPIB (C2), UBC (C3) and HPRT-1 (C4) In terms of relative expression levels, UBC is the highest, PPIB is considered a moderate-high, HPRT-1 and POLR2A are moderate-to low expressor target. | | 321808 - RNAscope 4-plex LS Multiplex Control Positive Probe -Hs | 321808 | 321818 - RNAscope 4-plex LS Multiplex Control Positive Probe -Mm | 321818 | 321828 - RNAscope 4-plex LS Multiplex Control Positive Probe -Rn | 321828 |
| |
RNAscope LS 2.5 Negative Control Probes | | RNAscope 3-plex LS Multiplex Negative Control Probe dapB in all 3 channels | 320878 | RNAscope 4-plex LS Multiplex Negative Control Probe dapB in all 4 channels | 321838 |
| |
RNAscope Control Slides (optional) Human Cell Pellet or Mouse 3T3 Cell Pellet | 310045 310023 | |
Order the following components:
| Component | Product Number | Product Description |
|---|
TSA Plus Fluorescein System for 50-150 Slides ACD Multiple TSA-F1 | NEL741001KT NEL741B001KT | |
TSA Plus Cyanine 3 System for 50-150 Slides ACD Multiple TSA-F2 | NEL744001KT NEL744B001KT | |
TSA Plus Cyanine 5 System for 50-150 Slides ACD Multiple TSA-F3 | NEL745001KT NEL745B001KT | |
TSA Vivid Fluorophore 520 | 323271 | TSA Vivid™ reagents are exceptionally bright dyes that offer an effective way to boost signal intensity and detect low-abundance targets in spatial biology applications. Contains 1x vial TSA Vivid fluorophore 520 and 1x vial DMSO |
TSA Vivid Fluorophore 570 | 323272 | TSA Vivid™ reagents are exceptionally bright dyes that offer an effective way to boost signal intensity and detect low-abundance targets in spatial biology applications. Contains 1x vial TSA Vivid fluorophore 570 and 1x vial DMSO |
TSA Vivid Fluorophore 650 | 323273 | TSA Vivid™ reagents are exceptionally bright dyes that offer an effective way to boost signal intensity and detect low-abundance targets in spatial biology applications. Contains 1x vial TSA Vivid fluorophore 650 and 1x vial DMSO |
| Opal 520 Reagent Pack | FP1487001KT | |
| Opal 570 Reagent Pack | FP1488001KT | |
| Opal 620 Reagent Pack | FP1495001KT | |
| Opal 690 Reagent Pack | FP1497001KT | |
Recommended dilution range for all reagents above: 1:750 - 1:3,000
Order the following components from Leica Biosystems (LeicaBiosystems.com):
| Component | Cat. No. |
|---|
| Bond Research Detection System | DS9455 |
| BOND Open Containers 30 mL | Op309700 |
| BOND Universal Covertiles 100 pack | S21.2001 |
| BOND Epitope Retrieval Solution 1-1L (RTU) | AR9961 |
| BOND Epitope Retrieval Solution 2-1L (RTU) | AR9640 |
| BOND Dewax Solution – 1L (RTU) | AR9222 |
| BOND Wash Solution 10X Concentrate – 1L | AR9590 |
| BOND Aspirating Probe Cleaning System | CS9100 |
| BOND Mixing Stations | S21.1971 |
