Learn more about our New BaseScopeTM assay

RNAscope® LS Multiplex Fluorescent Assay

Designed as an automated assay on the Leica Biosystems' BOND RX Research Advanced Staining System, this new assay enables single-molecule detection of up to four RNA targets simultaneously. Ideal for co-localization and co-expression studies, the assay and RNAscope® LS 2.5 Probes are available for application in your own lab and via ACD's Pharma Assay Services.  

The key technological advantage of this assay is the significant signal boost with simultaneous background noise suppression which is due to advances in ACD’s patented signal amplification technology combined with Perkin Elmer'sTyramide Signal Amplification (TSA) technology. Furthermore the signal sensitivity is balanced across all three channels.  These technological advances enable

  • Strong detection of low expressors-- Vivid visualization of even very low expressing genes in any of the three probe channels
  • Compatible with multiple sample types-- Detection of signals at low exposure and supports of FFPE samples and fresh frozen samples
  • Supports dual ISH and IHC protocols--High and stable signals enable sequential detection of proteins by IHC on the same samples. See Dual ISH & IHC for protocols.
  • Easy to use-- No need to match expression level of targets with channel sensitivity
  • Sharper, crisper signal-- Strong balanced signals across all three channels make it easy to generate publication-quality data.

 

"I was very impressed with the quality of work and quick turn-around of the Pharma Assay Services group.   The data generated using RNAscope LS Multiplex kit gave us enough information to proceed further with our study.  It allowed us to co-localize our target RNA along with different cell type specific RNA markers to determine in which CNS cell types the target was expressed.”

Curt Mazur

Director, Neuroscience Drug Discovery; Ionis Pharmaceuticals, Inc.

Sample Images
Lung cancer FFPE(RNA in situ hybridization ISH)

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

Expression of Hs-IDO1 (green), Hs-CD274 (PDL1)(red) and Hs-CTLA4 (white)in human lung cancer FFPE tissue using RNAscope LS multiplex Fluorescent assay(RNA in situ hybridization ISH)

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 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 CXCL13 (green), CD274 (PDL1) (red) and KRT`9-C3 (white) in human lung cancer FFPE tissue using RNAscope LS Multiplex Fluorescent Assay

Expression of Hs-TNFSF4 (OX40L)(green), Hs-CD274 (PDL1) (red) and Hs-HAVCR2 (TIM3) (white)in human lung cancer FFPE tissue using RNAscope LS multiplex Fluorescent assay(RNA in situ hybridization ISH)

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 CD8A (green) CD274 (red) combined with IHC of CD8 (white) using RNAscope LS multiplex Fluorescent assay in human lung cancer FFPE sample(RNA in situ hybridization ISH)

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

Expression of Hs-KRT19(green), Hs-PECAM1 (red) and Hs-MKI67(white) in human breast cancer FFPE tissue using RNAscope LS multiplex Fluorescent assay(RNA in situ hybridization ISH)

RNA expression of KRT19(green), PECAM1 (red) and MKI67(white) in human breast cancer FFPE tissue using RNAscope LS Multiplex Fluorescent Assay

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 in situ hybridization ISH)

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

Expression of Mm-Sc17a6 (Vglut2)(green), Mm-SLc17a7 (Vlgut1) (red) and Mm-Slc32a1 (white) in mouse brain fresh frozen tissue using RNAscope LS multiplex Fluorescent assay(RNA in situ hybridization ISH)

RNA expression of Sc17a6 (Vglut2)(green), SLc17a7 (Vlgut1) (red) and Slc32a1 (white) in mouse brain fresh frozen tissue using RNAscope LS Multiplex Fluorescent Assay

Expression of Mm-Cnr1(green), Mm-Drd1 (red) and Mm-Nmdar1 (white) in mouse brain FFPE tissue using RNAscope LS multiplex Fluorescent assay(RNA in situ hybridization ISH)

RNA expression of Cnr1(green), Drd1 (red) and Nmdar1 (white) in mouse brain FFPE tissue using RNAscope LS Multiplex Fluorescent Assay

Expression of Mm-Sc17a6-C1 (Vglut2) (green), Mm-Slc17a7-C2 (Vlgut1) (red) and Mm-Slc32a1-C3 (Vgat)(RNA in situ hybridization ISH)

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 ISH Expression of CD3D (green) CD8A (red) combined with IHC of CDC (white) using RNAscope LS multiplex Fluorescent assay in FFPE human tonsil(RNA in situ hybridization ISH)

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

Positive control probes demonstrate expression of Tbp (green), PPib (red) and UBC (white) in Mouse Brain Fresh Frozen(RNA in situ hybridization ISH)

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

Expression of MALAT1 (red) and Mog (white) using RNAscope LS multiplex Fluorescent assay(RNA in situ hybridization ISH)

RNA expression of Malat1 (red, high in nuclear) and Mog (white) in fresh frozen rat brain tissue using RNAscope LS multiplex Fluorescent Assay.

Expression of Hs-TNFSF4 (OX40L) (green), CD274 (PDL1) (red) and HAVCR2 (TIM3) (white) using RNAscope LS multiplex Fluorescent assay (RNA in situ hybridization ISH)

RNA expression of TNFSF4 (OX40L) (green), CD274 (PDL1) (red) and HAVCR2 (TIM3) (white) 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 FFPI 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 fluorophore3-plex filter4-plex fluorophore 4-plex filter

Fluorescein

Cyanine 3

Cyanine 5

 

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 (eg Nuance FX Multiplex Biomarker Imaging System)

Order the following components from ACD:

ComponentCat. No.

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 Flourescent 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. 

323120

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- HeLa Cells 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 from Perkin Elmer (perkinelmer.com):

ComponentProduct Number

TSA Plus Fluorescein System for 50-150 Slides

ACD Mulitple TSA-F1

NEL741001KT

NEL741B001KT

TSA Plus Cyanine 3 System for 50-150 Slides

ACD Mulitple TSA-F2

NEL744001KT

NEL744B001KT

TSA Plus Cyanine 5 System for 50-150 Slides

ACD Mulitple TSA-F3

NEL745001KT

NEL745B001KT

Opal 7-color fIHC Kit for 50-250 Slides

NEL797001KT

NEL797B001KT

Recommended dilution range for all reagents above: 1:750 - 1:3,000 

Order the following components from Leica Biosystems (LeicaBiosystems.com):

ComponentCat. No.
Bond Research Detection SystemDS9455
BOND Open Containers 30 mLOp309700
BOND Universal Covertiles 100 packS21.2001
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 – 1LAR9590
BOND Aspirating Probe Cleaning SystemCS9100
BOND Mixing Stations S21.1971

 

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