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RNAscope™, BaseScope™ and miRNAscope™ Probes

Aperio RNA ISH Algorithm by Leica Biosystems

Manual RNA ISH interpretation is time-consuming, subject to inter/intra-observer variability and typically employs semi-quantitative reads. The Aperio RNA ISH Algorithm from Leica Biosystems enables accurate counting of individual signals across the tissue, providing standardized, reproducible results. Validation testing has demonstrated the high correlation between the Aperio RNA ISH Algorithm and expert manual review, making it ideal for researchers who want to automate routine counting tasks.

Aperio RNA ISH Algorithm Features:

Aperio RNA ISH Algorithm is compatible with the chromogenic RNAscope Manual and Automated Assays:

RNAscope™ 2.0 HD Reagent Kit—BROWN
RNAscope™ 2.0 HD Reagent Kit—RED
RNAscope™ 2.5 HD Reagent Kit—BROWN
RNAscope™ 2.5 HD Reagent Kit—RED
RNAscope™ 2-Plex Reagent Kit
RNAscope™ LS Reagent Kit—BROWN
RNAscope™ LS Reagent Kit—RED
RNAscope™ VS Reagent Kit—BROWN
RNAscope™ VS Reagent Kit—RED

The Aperio RNA ISH Algorithm forms part of Leica Biosystems’ comprehensive and open platform for ISH research, including the BOND RX rapid staining system, Aperio digital pathology scanners, and powerful software for image management and analysis. For more information, visit LeicaBiosystems.com/Aperio.

Aperio RNA ISH Algorithm and Leica Biosystems BOND RX are: For Research Use Only. Not For Use In Diagnostic Procedures.

LEICA and the Leica logo are registered trademarks of Leica Microsystems IR GmbH. Aperio and BOND are trademarks of the Leica Biosystems group of companies in the USA and optionally in other countries. Other logos, product and/or company names might be trademarks of their respective owners. 95.14542 Rev A 12/2016

For more information contact us at LeicaBiosystems.com.

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 Professional Assay Services.

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

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

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Control Probes for Manual Assays: RNAscope 2.5 HD Duplex Reagent Kit

Species-specific positive probes and negative control probes are required to run with human or mouse control slides for the technical quality control check and with your sample for Sample or RNA quality control check. ACD provides the positive control probes that target species-specific housekeeping genes and negative control probes that target the bacteria DapB gene.

When working with RNAscope™ 2.5 HD Duplex chromogenic assay, you will need one premixed duplex positive control probe and one premixed duplex negative control probe.

RNAscope™ 2.5 Duplex Positive Control Probe

Human (Cat. No. 321641), Mouse (Cat. No. 321651)

Each control probe set contains premixed and ready to use PPIB probe in channel 1 and POLR2A probe in channel 2. Probe volume is configured for 20 reactions.

RNAscope™ 2-plex Negative Control Probe

(Cat. No. 320751)

Contains premixed probe for DapB in both channel 1 and 2. Probe volume is configured for 20 reactions.

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RNAscope™ 2.5 HD Duplex Assay

The RNAscope 2.5 HD Duplex Assay is based on ACD’s patented signal amplification and background suppression technology. The assay uses a novel and proprietary method of in situ hybridization (ISH) to visualize single RNA molecules per cell in FFPE samples mounted on slides. RNAscope Assays do not require the RNA-free environment used for traditional ISH. RNAscope probes in Channel 1 and Channel 2 are compatible with this assay which utilizes HRP-based Green and AP-based Fast Red chromogens to result in a detectable green and red signal respectively.

RNAscope 2.5 HD Duplex Reagent Kit gives reliable results which are ideal for archiving due to the permanent staining visible under any standard bright field microscope. The RNAscope 2.5 HD Duplex Reagent Kits are designed to provide more intense staining when low copy target gene expression is anticipated (1–20 copies per cell). The RNAscope 2.5 HD Duplex reagent kits are compatible with the same RNAscope probes used previously with RNAscope 2.0 2-plex assays.

For customers using RNAscope 2.5 HD Duplex Manual Assay for the first time, ACD's Intro Packs include species-specific control slides, control probes, the Reagent Kit, the ImmEdge hydrophobic barrier pen and the must-have accessory VectaMount™ Mounting Medium from Vecta Labs. Be sure to order your target C1 and C2 probes separately.

The RNAscope 2.5 HD Duplex Manual Assay Workflow Requires:

RNAscope Target Probes (Catalog or Made-to-Order C1 & C2 Probes, compatitle with the same RNAscope probes used previously with RNAscope 2.0 2-plex assays.)
RNAscope Duplex Positive Control Probes
RNAscope Duplex Negative Control Probes (Cat. No. 320751)
RNAscope Control Slides (optional)
RNAscope 2.5 HD Duplex Reagent Kit (Cat. no 322430) (Includes Pretreatment Kit for FFPE samples, detection kit and wash buffer)
Vector Labs Vectamount Permanent Mounting Medium (Cat. No. 321584)
ImmEdge Hydrophobic Barrier Pen (Cat. No 310018)
HybEZ™ Hybridization System
Brightfield microscope

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RNAscope™ 2.5 LS Duplex Assay

The RNAscope™ 2.5 LS Duplex Assay is designed as an automated assay on the Leica Biosystems' BOND RX Research Advanced Staining System. The RNAscope 2.5 LS Duplex Assay, is an RNA in situ hybridization assay based on ACD’s patented signal amplification and background suppression technology. The assay uses a novel and proprietary method of in situ hybridization (ISH) to visualize single RNA molecules per cell in a multitude of sample types mounted on slides. RNAscope Assays do not require the RNA-free environment used for traditional ISH.

The RNAscope 2.5 LS Automated Assay requires the following from Leica Biosystems (LeicaBiosystems.com):

Component	Cat. No.
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 Polymer Refine Detection (DAB) and Hematoxylin^*	DS9800
BOND Polymer Refine Red Detection*	DS9390
BOND DAB Enhancer 30ml	AR9432
BOND Aspirating Probe Cleaning System	CS9100
BOND Mixing Stations	S21.1971

RNAscope 2.5 LS Duplex Reagent Kit Workflow Requires the following from ACD:

RNAscope 2.5 LS Target Probes (Catalog or Made-to-Order LS Probes)
300038 RNAscope 2.5 LS Probe (RTU)
300038-C2 RNAscope 2.5 LS Probe- C2 (50x)
RNAscope 2.5 LS Control Probes choose from Pooled Control Probes for both C1 and C2 channels or Control probes in C2 only:
- Pooled Control Probes

320768 RNAscope 2.5 LS Duplex Control Probes (PPIB-C1, Polr2A-C2)- Mm

320748 RNAscope 2.5 LS Duplex Control Probes (PPIB-C1, Polr2A-C2)- Hu

320758 RNAscope 2.5 LS Duplex Negative Control Probe (DapB-C1, DapB-C2)

Control Probes for C2 only (to be used with C1 RTU Target probes)

313908-C2 RNAscope 2.5 LS Positive Control Probe - Hs-PPIB-C2 (50X)

312038-C2 RNAscope 2.5 LS Negative Control Probe - DapB-C2 (50x)

RNAscope Control Slides (optional)
- Human Cell Pellet Cat. No. 310045, or Mouse 3T3 Cell Pellet Cat. No. 310023
RNAscope 2.5 LS Duplex Reagent Kit Cat No. 322440
RNAscope 2.5 LS Green Accessory Pack Cat No. 322550

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RNAscope™, BaseScope™ and miRNAscope™ Probes

Aperio RNA ISH Algorithm by Leica Biosystems

RNAscope™ LS Multiplex Fluorescent Assay

Technology Overview

Guidelines for Microscope Channel Selection

What to order

Control Probes for Manual Assays: RNAscope 2.5 HD Duplex Reagent Kit

RNAscope™ 2.5 HD Duplex Assay

RNAscope™ 2.5 LS Duplex Assay

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