Getting started with RNAscope

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Getting Started with RNAscope® Assays What’s Inside

1. Optimization Steps

2. Pretreatment Guidelines

3. User Manual and Reagent Selection

4. Product Overview

Get started today at acdbio.com/go

2 RNAscope® Getting Started Brochure

Before you begin Follow our recommended procedures for your specific tissue type and detection assay. Before you begin with the RNAscope® or BaseScope™ assay, please refer to the support website, for the following links:

Download Download the user manual based on the selection guide table found at acdbio.com/go

Select Select your RNAscope® pretreatment reagents based on your tissue sample preparation method

Qualify Qualify your samples using control probes and run your target probes

3RNAscope® Getting Started Brochure

Optimize your assay, get better results

Always use control probes and slides

Run the RNAscope® assay with the control slides to validate the assay workflow and use control probes on your tissue sample to validate the quality of RNA in your samples. The housekeeping gene PPIB (Cyclophilin B), is commonly used as a positive control. The negative control probes target the bacteria DapB gene.

Optimize RNAscope® assay conditions for your tissues to obtain optimal data

Target Retrieval and Protease conditions might require optimization depending on tissue samples type. Further optimization might be required if samples are not prepared according to ACD’s recommendations: for FFPE samples, for 16-32 hours at room temperature in fresh 10% neutral buffered formalin (NBF) or 4% paraformaldehyde (PFA).

Check that all required materials are available Download the RNAscope® Manual Assay Pre-Run Checklist by following this link: acdbio.com/go

4 RNAscope® Getting Started Brochure

Please follow the appropriate user manuals or technical notes to define the pretreatment conditions for your tissue sample and tissue type. Where the sample type is FFPE, start with the standard conditions of 15 minutes incubation with Target Retrieval, and 30 minutes incubation with Protease Plus. Pretreatment conditions may need adjustment based on initial results (cell morphology/signal quality).

As a first step, vary Target Retrieval incubation time (example: for over‑fixed tissues you can start by varying from 15 to 30 minutes). This may need to be decreased for under‑fixed tissues or increased for over‑fixed tissues, depending on the observed staining pattern (see Figure 1).

If the results are still suboptimal, modify Protease Plus time. Protease Plus may need to be decreased for under‑fixed tissues or increased for over‑fixed tissues, depending on the observed staining pattern (example: for under‑fixed tissues you can start by varying from 30 to 15 minutes) (see Figure 2).

Use the optimized conditions for the samples with all target probes. For fixed/fresh frozen tissues you may need to vary the protease step.

Downloads RNAscope® Reference Guide: acdbio.com/referenceguide/ RNAscope® Data Analysis Guide: acdbio.com/dataanalysisguide/

Pretreatment guidelines for FFPE samples

5RNAscope® Getting Started Brochure

Target Retrieval Figure 1. Target Retrieval incubation time scale

Under‑fixed† or special sample/tissue types*

0 5 10 15 20 25 30 35 40 45

Over‑fixed†† or special sample/tissue types**

Standard Time (min)

Note: ACD recommends fixing cells for FFPE cell pellets preparation with 10% formaldehyde for 24 hours. Prepare 10% formaldehyde with 37% formaldehyde, 10X PBS, and distilled water (e.g to make 100 mL of 10% formaldehyde, add 27 mL 37% formaldehyde and 10 mL 10X PBS to 63 mL water). Using 1X PBS instead of 10X PBS can cause changes in fixation conditions and affect assay results.

Protease Plus Figure 2. Protease Plus incubation time scale

Standard Time (min)

Under‑fixed† or special sample/tissue types*

0 5 10 15 20 25 30 35 40 45

Over‑fixed†† or special sample/tissue types**

† Relative to RNAscope® standards, tissue might be under-fixed when using the following conditions:

†† Relative to RNAscope® standards, tissue is considered over-fixed when using the following conditions:

*Special tissue samples: **Special tissue samples:

4% PFA or 10% NBF <24 hours, 4°C or RT

4% PFA or 10% NBF >24 hours, 4°C or RT

For tissues like xenografts, lymphoid tissues (spleen, tonsil, lymph node, thymus, lymphoma), or retina: please refer to the RNAscope® Reference Guide: acdbio.com/referenceguide

For cultured cells samples: please refer to table on page 8&9.

For tissues like muscle, kidney or liver please refer the RNAscope® Reference Guide: acdbio.com/ referenceguide

Note: The volume provided is the volume required to perform 20 slides. Based on the density and viscosity of each reagent, the drop size volume is not the same and therefore each reagent is provided in different volumes (based on the number of drops).

6 RNAscope® Getting Started Brochure

Sample Type RNAscope®/BaseScope™ Detection Assay RNAscope® Pretreatment Reagents Sample Preparation Part 1 User Guide/Tech Note

RNAscope®/BaseScope™ Detection Reagent Part 2 User Manual

FFPE Formalin-Fixed Paraffin‑ Embedded

2.5 HD Chromogenic BROWN, RED, Duplex RNAscope® Hydrogen Peroxide, Target Retrieval, and Protease Plus Formalin‑Fixed Paraffin‑Embedded (FFPE) Sample Preparation and Pretreatment PART 1 Doc. No. 322452 Preparing FFPE cell pellets for the RNAscope® and BaseScope™ assays SOP 45-008

For BROWN: RNAscope® 2.5 HD Detection Reagent –BROWN, User Manual PART 2 Doc. No. 322310-USM For RED: RNAscope® 2.5 HD Detection Reagent –RED, User Manual PART 2 Doc. No. 322360-USM

For Duplex: RNAscope® 2.5 HD Duplex Detection Kit (Chromogenic), User Manual PART 2 Doc. No. 322500-USM

Multiplex Fluorescent v2 RNAscope® Hydrogen Peroxide, Target Retrieval, and Protease Plus RNAscope® Multiplex Fluorescent Reagent Kit v2 User Manual with Sample Preparation and Pretreatment Doc. No. 323100-USM Preparing FFPE cell pellets for the RNAscope® and BaseScope™ assays SOP 45-008

RNAscope® Multiplex Fluorescent Reagent Kit v2 User Manual with Sample Preparation and Pretreatment Doc. No. 323100-USM When running Multiplex Fluorescent v2 4-plex assay: RNAscope® Multiplex Fluorescent Reagent Kit v2 User Manual with Sample Preparation and Pretreatment Doc. No. 323100- USM With RNAscope® 4-plex Ancillary Kit for Multiplex Fluorescent Reagent Kit v2 Tech Note Cat. No. 323120

BaseScope™ RED, BaseScope™ RED v2, BaseScope™ Duplex

RNAscope® Hydrogen Peroxide, Target Retrieval, and Protease III/IV For BaseScope™ RED: BaseScope™ Detection Reagent Kit RED User Manual with FFPE Sample Preparation and Pretreatment Doc. No. 322900-USM Preparing FFPE cell pellets for the RNAscope® and BaseScope™ assays SOP 45-008

For BaseScope™ Duplex: BaseScope™ Duplex Detection Reagent Kit User Manual with FFPE Sample Preparation and Pretreatment Doc. No. 323800-USM Preparing FFPE cell pellets for the RNAscope® and BaseScope™ assays SOP 45-008

For BaseScope™ RED: BaseScope™ Detection Reagent Kit RED User Manual with FFPE Sample Preparation and Pretreatment Doc. No. 322900-USM For BaseScope™ RED v2: BaseScope™ Detection Reagent Kit RED User Manual with FFPE Sample Preparation and Pretreatment Doc. No. UM 323900 For BaseScope™ Duplex: BaseScope™ Duplex Detection Reagent Kit User Manual with FFPE Sample Preparation and Pretreatment Doc. No. 323800-USM

Fresh Frozen 2.5 HD Chromogenic Brown/Red, Duplex RNAscope® Hydrogen Peroxide, Protease IV** Preparing Fresh Frozen Tissue for the RNAscope® and BaseScope™ Assays Tech Note Cat. No. MK 50-013 Sample Preparation Technical Note for Fresh Frozen Tissue Using RNAscope® 2.5 Chromogenic Assay (Single-plex and Duplex) Tech Note Cat. No 320536

For 2.5 HD Chromogenic BROWN: RNAscope® 2.5 HD Detection Reagent –BROWN User Manual PART 2 Doc. No.322310-USM

For 2.5 HD Chromogenic RED: RNAscope® 2.5 HD Detection Reagent –RED User Manual PART 2 Doc. No.322360-USM

For 2.5 HD Chromogenic Duplex: RNAscope® 2.5 HD Duplex Detection Kit (Chromogenic) User Manual PART 2 Doc. No. 322500-USM

Multiplex Fluorescent RNAscope® Protease IV Preparing Fresh Frozen Tissue for the RNAscope® and BaseScope™ Assays Tech Note Cat. No. MK 50-013

RNAscope® Sample Preparation and Pretreatment Guide for Fresh Frozen Tissue (For Manual RNAscope® Assay) PART 1 Doc. No. 320513

RNAscope® Fluorescent Multiplex Reagent Kit User Manual PART 2 Doc. No. 320293

Multiplex Fluorescent v2 RNAscope® Hydrogen Peroxide, Protease IV Preparing Fresh Frozen Tissue for the RNAscope® and BaseScope™ Assays Tech Note Cat. No. MK50-013 RNAscope® Multiplex Fluorescent Reagent Kit v2 User Manual with Sample Preparation and Pretreatment Doc. No. 323100-USM

RNAscope® Multiplex Fluorescent Reagent Kit v2 User Manual with Sample Preparation and Pretreatment Doc. No. 323100-USM When running Multiplex Fluorescent v2 4-plex assay: RNAscope® Multiplex Fluorescent Re- agent Kit v2 User Manual with Sample Preparation and Pretreatment Doc. No. 323100-USM With RNAscope® 4-plex Ancillary Kit for Multiplex Fluorescent Reagent Kit v2 Tech Note Cat. No. 323120

BaseScope™ RED, BaseScope™ RED v2, BaseScope™ Duplex

RNAscope® Hydrogen Peroxide, Protease IV Preparing Fresh Frozen Tissue for the RNAscope® and BaseScope™ Assays Tech Note Cat. No. MK50-013

Sample Preparation Technical Note for Fresh Frozen Tissue Using RNAscope® 2.5 Chromogenic Assay (Single-plex and Duplex) Tech Note Cat. No 320536

Fixed Frozen 2.5 HD Chromogenic Brown/Red, Duplex RNAscope® Hydrogen Peroxide, Target Retrieval and Protease Plus Sample Preparation Technical Note for Fixed Frozen Tissue Using RNAscope® 2.5 Chromogenic Assay (Single-plex and Duplex) Tech Note Cat. No. 320534

For 2.5 HD Chromogenic BROWN: RNAscope® 2.5 HD Detection Reagent –BROWN User Manual PART 2 Doc. No. 322310-USM For 2.5 HD Chromogenic RED: RNAscope® 2.5 HD Detection Reagent –RED User Manual PART 2 Doc. No. 322360-USM For 2.5 HD Chromogenic Duplex: RNAscope® 2.5 HD Duplex Detection Kit (Chromogenic) User Manual PART 2 Doc. No. 322500-USM

User manual and reagent selection guide

7RNAscope® Getting Started Brochure

Sample Type RNAscope®/BaseScope™ Detection Assay RNAscope® Pretreatment Reagents Sample Preparation Part 1 User Guide/Tech Note

RNAscope®/BaseScope™ Detection Reagent Part 2 User Manual

FFPE Formalin-Fixed Paraffin‑ Embedded

For BROWN: RNAscope® 2.5 HD Detection Reagent –BROWN, User Manual PART 2 Doc. No. 322310-USM For RED: RNAscope® 2.5 HD Detection Reagent –RED, User Manual PART 2 Doc. No. 322360-USM

For Duplex: RNAscope® 2.5 HD Duplex Detection Kit (Chromogenic), User Manual PART 2 Doc. No. 322500-USM

BaseScope™ RED, BaseScope™ RED v2, BaseScope™ Duplex

For 2.5 HD Chromogenic BROWN: RNAscope® 2.5 HD Detection Reagent –BROWN User Manual PART 2 Doc. No.322310-USM

For 2.5 HD Chromogenic RED: RNAscope® 2.5 HD Detection Reagent –RED User Manual PART 2 Doc. No.322360-USM

For 2.5 HD Chromogenic Duplex: RNAscope® 2.5 HD Duplex Detection Kit (Chromogenic) User Manual PART 2 Doc. No. 322500-USM

Multiplex Fluorescent RNAscope® Protease IV Preparing Fresh Frozen Tissue for the RNAscope® and BaseScope™ Assays Tech Note Cat. No. MK 50-013

RNAscope® Sample Preparation and Pretreatment Guide for Fresh Frozen Tissue (For Manual RNAscope® Assay) PART 1 Doc. No. 320513

RNAscope® Fluorescent Multiplex Reagent Kit User Manual PART 2 Doc. No. 320293

RNAscope® Multiplex Fluorescent Reagent Kit v2 User Manual with Sample Preparation and Pretreatment Doc. No. 323100-USM When running Multiplex Fluorescent v2 4-plex assay: RNAscope® Multiplex Fluorescent Re- agent Kit v2 User Manual with Sample Preparation and Pretreatment Doc. No. 323100-USM With RNAscope® 4-plex Ancillary Kit for Multiplex Fluorescent Reagent Kit v2 Tech Note Cat. No. 323120

BaseScope™ RED, BaseScope™ RED v2, BaseScope™ Duplex

RNAscope® Hydrogen Peroxide, Protease IV Preparing Fresh Frozen Tissue for the RNAscope® and BaseScope™ Assays Tech Note Cat. No. MK50-013

Sample Preparation Technical Note for Fresh Frozen Tissue Using RNAscope® 2.5 Chromogenic Assay (Single-plex and Duplex) Tech Note Cat. No 320536

8 RNAscope® Getting Started Brochure

Sample Type RNAscope®/BaseScope™ Detection Assay RNAscope® Pretreatment Reagents

Sample Preparation Part 1 User Guide/Tech Note

RNAscope®/BaseScope™ Detection Reagent Part 2 User Manual

Fixed Frozen Multiplex Fluorescent RNAscope® Target Retrieval**, Protease III Preparing Fixed Frozen Tissue for the RNAscope® Fluorescent Multiplex Assay Tech Note Cat. No. 320535

RNAscope® Fluorescent Multiplex Reagent Kit User Manual PART 2 Doc. No. 320293

Multiplex Fluorescent v2 RNAscope® Hydrogen Peroxide, Target Retrieval and Protease III RNAscope® Multiplex Fluorescent Reagent Kit v2 User Manual with Sample Preparation and Pretreatment Doc. No. 323100-USM

RNAscope® Multiplex Fluorescent Reagent Kit v2 User Manual with Sample Preparation and Pretreatment Doc. No. 323100-USM When running Multiplex Fluorescent v2 4-plex assay: RNAscope® Multiplex Fluorescent Re- agent Kit v2 User Manual with Sample Preparation and Pretreatment Doc. No. 323100-USM With RNAscope® 4-plex Ancillary Kit for Multiplex Fluorescent Reagent Kit v2 Tech Note Cat. No. 323120

BaseScope™ RED, BaseScope™ RED v2, BaseScope™ Duplex*

RNAscope® Hydrogen Peroxide, Target Retrieval,Protease III/IV Sample Preparation Technical Note for Fixed Frozen Tissue Using RNAscope® 2.5 Chromogenic Assay (Single-plex and Duplex) Tech Note Cat. No. 320534

Adherent / Cultured Cells 2.5 HD Chromogenic Brown/Red, Duplex RNAscope® Hydrogen Peroxide, Protease III** Sample Preparation Technical Note for Cultured Adherent Cells Using RNAscope® 2.5 Chromogenic Assay (Single-plex and Duplex) Tech Note Cat. No. 321232 Optional: RNAscope® Assay for Adherent Cells Cultured on Covers- lips Tech Note Cat. No. MK 50-012

For 2.5 HD Chromogenic BROWN: RNAscope® 2.5 HD Detection Reagent –BROWN, User Manual PART 2 Doc. No. 322310-USM

For 2.5 HD Chromogenic RED: RNAscope® 2.5 HD Detection Reagent –RED, User Manual PART 2 Doc. No. 322360-USM

For 2.5 HD Chromogenic Duplex: RNAscope® 2.5 HD Duplex Detection Kit (Chromogenic), User Manual PART 2 Doc. No. 322500-USM

Multiplex Fluorescent RNAscope® Protease III Sample Preparation Technical Note for Cultured Adherent Cells Using RNAscope® Fluorescent Assay Tech Note Cat. No. 320538 Optional: RNAscope® Assay for Adherent Cells Cultured on Covers- lips Tech Note Cat. No. MK 50-012

RNAscope® Fluorescent Multiplex Reagent Kit User Manual PART 2 Doc. No. 320293

Multiplex Fluorescent v2 RNAscope® Hydrogen Peroxide, Protease III Cultured Adherent Cell Sample Preparation for the RNAscope® Multiplex Fluorescent Assay v2 Tech Note Cat. No. MK 50 010 Optional: RNAscope® Assay for Adherent Cells Cultured on Covers- lips Tech Note Cat. No. MK 50-012

RNAscope® Multiplex FluorescentReagent Kit v2, User Manual with Sample Preparation and Pretreatment Doc. No. 323100-USM When running Multiplex Fluorescent v2 4-plex assay: RNAscope® Multiplex Fluorescent Re- agent Kit v2 User Manual with Sample Preparation and Pretreatment Doc. No. 323100-USM With RNAscope® 4-plex Ancillary Kit for Multiplex Fluorescent Reagent Kit v2 Tech Note Cat. No. 323120

BaseScope™ RED, BaseScope™ RED v2, BaseScope™ Duplex

RNAscope® Hydrogen Peroxide, Protease III or IV Sample Preparation Technical Note for Cultured Adherent Cells Using RNAscope® 2.5 Chromogenic Assay (Single-plex and Duplex) Tech Note Cat. No.321232 Optional: RNAscope® Assay for Adherent Cells Cultured on Cov- er-slips Tech Note Cat. No. MK 50-012

Non-Adherent Cells 2.5 HD Chromogenic Brown/Red, Duplex RNAscope® Hydrogen Peroxide, Protease III** Sample Preparation Technical Note for Non-Adherent Cells using RNAscope® 2.5 and BaseScope™ Chromogenic Assays. Tech Note TS 46-004

For 2.5 HD Chromogenic BROWN: RNAscope® 2.5 HD Detection Reagent –BROWN User Manual PART 2 Doc. No. 322310-USM

For 2.5 HD Chromogenic RED: RNAscope® 2.5 HD Detection Reagent –RED User Manual PART 2 Doc. No. 322360-USM

For 2.5 HD Chromogenic Duplex: RNAscope® 2.5 HD Duplex Detection Kit (Chromogenic) User Manual PART 2 Doc. No. 322500-USM

Multiplex Fluorescent RNAscope® Protease III Sample Preparation Technical Note Non-Adherent Cells for the RNAscope® Fluorescent Multiplex Assay Tech Note TS 46-005

RNAscope® Fluorescent Multiplex Reagent Kit User Manual PART 2 Doc. No. 320293

Multiplex Fluorescent v2* RNAscope® Hydrogen Peroxide, Protease III Sample Preparation Technical Note for Non-Adherent Cells using RNAscope® 2.5 and BaseScope™ Chromogenic Assays. Tech Note TS 46-004

RNAscope® Multiplex FluorescentReagent Kit v2 User Manual with Sample Preparation and Pretreatment Doc. No. 323100-USM When running Multiplex Fluorescent v2 4-plex assay: RNAscope® Multiplex Fluorescent Re- agent Kit v2 User Manual with Sample Preparation and Pretreatment Doc. No. 323100-USM With RNAscope® 4-plex Ancillary Kit for Multiplex Fluorescent Reagent Kit v2 Tech Note Cat. No. 323120

User manual and reagent selection guide (continued)

9RNAscope® Getting Started Brochure

Sample Type RNAscope®/BaseScope™ Detection Assay RNAscope® Pretreatment Reagents

Sample Preparation Part 1 User Guide/Tech Note

RNAscope®/BaseScope™ Detection Reagent Part 2 User Manual

Fixed Frozen Multiplex Fluorescent RNAscope® Target Retrieval**, Protease III Preparing Fixed Frozen Tissue for the RNAscope® Fluorescent Multiplex Assay Tech Note Cat. No. 320535

RNAscope® Fluorescent Multiplex Reagent Kit User Manual PART 2 Doc. No. 320293

RNAscope® Multiplex Fluorescent Reagent Kit v2 User Manual with Sample Preparation and Pretreatment Doc. No. 323100-USM When running Multiplex Fluorescent v2 4-plex assay: RNAscope® Multiplex Fluorescent Re- agent Kit v2 User Manual with Sample Preparation and Pretreatment Doc. No. 323100-USM With RNAscope® 4-plex Ancillary Kit for Multiplex Fluorescent Reagent Kit v2 Tech Note Cat. No. 323120

BaseScope™ RED, BaseScope™ RED v2, BaseScope™ Duplex*

For 2.5 HD Chromogenic BROWN: RNAscope® 2.5 HD Detection Reagent –BROWN, User Manual PART 2 Doc. No. 322310-USM

For 2.5 HD Chromogenic RED: RNAscope® 2.5 HD Detection Reagent –RED, User Manual PART 2 Doc. No. 322360-USM

For 2.5 HD Chromogenic Duplex: RNAscope® 2.5 HD Duplex Detection Kit (Chromogenic), User Manual PART 2 Doc. No. 322500-USM

RNAscope® Fluorescent Multiplex Reagent Kit User Manual PART 2 Doc. No. 320293

BaseScope™ RED, BaseScope™ RED v2, BaseScope™ Duplex

For 2.5 HD Chromogenic BROWN: RNAscope® 2.5 HD Detection Reagent –BROWN User Manual PART 2 Doc. No. 322310-USM

For 2.5 HD Chromogenic RED: RNAscope® 2.5 HD Detection Reagent –RED User Manual PART 2 Doc. No. 322360-USM

For 2.5 HD Chromogenic Duplex: RNAscope® 2.5 HD Duplex Detection Kit (Chromogenic) User Manual PART 2 Doc. No. 322500-USM

Multiplex Fluorescent RNAscope® Protease III Sample Preparation Technical Note Non-Adherent Cells for the RNAscope® Fluorescent Multiplex Assay Tech Note TS 46-005

RNAscope® Fluorescent Multiplex Reagent Kit User Manual PART 2 Doc. No. 320293

RNAscope® Multiplex FluorescentReagent Kit v2 User Manual with Sample Preparation and Pretreatment Doc. No. 323100-USM When running Multiplex Fluorescent v2 4-plex assay: RNAscope® Multiplex Fluorescent Re- agent Kit v2 User Manual with Sample Preparation and Pretreatment Doc. No. 323100-USM With RNAscope® 4-plex Ancillary Kit for Multiplex Fluorescent Reagent Kit v2 Tech Note Cat. No. 323120

10 RNAscope® Getting Started Brochure

Sample Type RNAscope®/BaseScope™ Detection Assay RNAscope® Pretreatment Reagents

Sample Preparation Part 1 User Guide/Tech Note

RNAscope®/BaseScope™ Detection Reagent Part 2 User Manual

Non-Adherent Cells BaseScope™ RED, BaseScope™ RED v2, BaseScope™ Duplex*

RNAscope® Hydrogen Peroxide, Protease III or IV Sample Preparation Technical Note for Non-Adherent Cells using RNAscope® 2.5 and BaseScope™ Chromogenic Assays. Tech Note TS 46-004

Whole-Mount 2.5 HD Chromogenic Red RNAscope® Target Retrieval, Protease Plus, probe diluent** RNAscope® Assay on Whole Zebrafish Embryos Tech Note Cat. No. MK 50-016

RNAscope® 2.5 HD Detection Reagent –RED User Manual PART 2 Doc. No. 322360-USM

Multiplex Fluorescent v2 RNAscope® Target Retrieval, Protease Plus, probe diluent** RNAscope® Assay on Whole Zebrafish Embryos Tech Note Cat. No. MK 50-016

RNAscope® Multiplex Fluorescent Reagent Kit v2, User Manual with Sample Preparation and Pretreatment Doc. No. 323100-USM When running 4-plex assay: RNAscope® 4-plex Ancillary Kit for Multiplex Fluorescent Reagent Kit v2 Tech Note Cat. No. 323120

Adherent / Cultured Cells 96well Plate

Multiplex Fluorescent RNAscope® Protease III RNAscope® Fluorescent Assay for Cultured Adherent Cells in 96- well Plate Format Tech Note

RNAscope® Fluorescent Multiplex Reagent Kit User Manual PART 2 Doc. No. 320293

Multiplex Fluorescent v2 RNAscope® Hydrogen Peroxide, Protease III RNAscope® Multiplex Fluorescent v2 Assay for Cultured Adherent Cells in 96-well Plate Format SOP 45-009A

RNAscope® Multiplex Fluorescent Reagent Kit v2 User Manual with Sample Preparation and Pretreatment Doc. No. 323100-USM When running 4-plex assay: RNAscope® 4-plex Ancillary Kit for Multiplex Fluorescent Reagent Kit v2 Tech Note Cat. No. 323120

For any other tissue/cell samples preparation, please contact support@acdbio.com * Not validated internally. ** This pretreatment buffer is not a component of the Reagent kit, it needs to be purchased separately.

User manual and reagent selection guide (continued)

Assay Type Sample Type RNAscope®/BaseScope™ Detection Assay

RNAscope®/BaseScope™ Detection Reagent User Manual

Leica BOND RX and Leica BOND RXm

FFPE Formalin-Fixed Paraffin‑Embedded

RNAscope® 2.5 LS Reagent Kit – BROWN, – RED, Duplex For 2.5 LS BROWN: RNAscope® 2.5 LS Reagent Kit – BROWN (BDZ9) User Manual Doc. No. UM-322100 RNAscope® 2.5 LS Reagent Kit – BROWN (BDZ11) User Manual Doc. No. 322100-USM

For 2.5 LS RED: RNAscope® 2.5 LS Reagent Kit – RED (BDZ9) User Manual Doc. No. UM-322150 RNAscope® 2.5 LS Reagent Kit – RED (BDZ11) User Manual Doc. No. 322150-USM

For 2.5 LS Duplex: RNAscope® 2.5 LS Duplex Reagent Kit (BDZ11) User Manual Doc. No. 322440-USM

RNAscope® LS Multiplex Fluorescent Assay RNAscope® LS Multiplex Fluorescent Reagent Kit (BDZ11) User Manual Doc. No. 322800-USM Performing automated 4-plex in situ hybridization (ISH) with the RNAscope® LS 4-plex Fluorescent Assay Tech Note Doc. No. 322380-TN

RNAscope® 2.5 LSx Reagent Kit – BROWN and – RED For 2.5 LS BROWN: RNAscope® 2.5 LSx Reagent Kit – BROWN (BDZ15/BXD15) User Manual Doc. No. UM-322700

For 2.5 LS RED: RNAscope® 2.5 LSx Reagent Kit – RED (BDZ15/BXD15) User Manual Doc. No. 322750-USM

BaseScope™ LS Reagent Kit – RED BaseScope™ LS Detection Reagent Kit (BDZ11 or higher) User Manual Doc. No. 323600-USM-ULT

Ventana DISCOVERY™ ULTRA System

FFPE Formalin-Fixed Paraffin‑Embedded

RNAscope® VS Universal HRP Assay (BROWN) RNAscope® VS Universal HRP Assay (BROWN) User Manual Doc. No. 323200-USM-ULT

RNAscope® VS Universal AP Assay (RED) RNAscope® VS Universal AP Assay (RED) User Manual Doc. No. 323250-USM-ULT

RNAscope® VS Duplex Assay (RED/BROWN) RNAscope® VS Duplex Assay (RED/BROWN) User Manual Doc. No. 323300-USM-ULT

BaseScope™ VS Assay (RED), BaseScope™ VS Assay (RED) User Manual Doc. No. 323700-USM-ULT

Ventana DISCOVERY XT™ System*

FFPE Formalin-Fixed Paraffin‑Embedded

RNAscope® 2.5 VS Assay (BROWN) RNAscope® 2.5 VS Assay (BROWN) User Manual Doc. No. 322200-USM-XT

RNAscope® 2.5 VS Assay (RED) RNAscope® 2.5 VS Assay (RED) User Manual Doc. No. 322250-USM-XT

User manual and reagent selection guide for automated assays

* Please note that the RNAscope® VS Duplex and the BaseScope™ assay are not available on the DISCOVERY XT

11RNAscope® Getting Started Brochure

Sample Type RNAscope®/BaseScope™ Detection Assay RNAscope® Pretreatment Reagents

Sample Preparation Part 1 User Guide/Tech Note

RNAscope®/BaseScope™ Detection Reagent Part 2 User Manual

Non-Adherent Cells BaseScope™ RED, BaseScope™ RED v2, BaseScope™ Duplex*

RNAscope® Hydrogen Peroxide, Protease III or IV Sample Preparation Technical Note for Non-Adherent Cells using RNAscope® 2.5 and BaseScope™ Chromogenic Assays. Tech Note TS 46-004

Whole-Mount 2.5 HD Chromogenic Red RNAscope® Target Retrieval, Protease Plus, probe diluent** RNAscope® Assay on Whole Zebrafish Embryos Tech Note Cat. No. MK 50-016

RNAscope® 2.5 HD Detection Reagent –RED User Manual PART 2 Doc. No. 322360-USM

Multiplex Fluorescent v2 RNAscope® Target Retrieval, Protease Plus, probe diluent** RNAscope® Assay on Whole Zebrafish Embryos Tech Note Cat. No. MK 50-016

Adherent / Cultured Cells 96well Plate

Multiplex Fluorescent RNAscope® Protease III RNAscope® Fluorescent Assay for Cultured Adherent Cells in 96- well Plate Format Tech Note

RNAscope® Fluorescent Multiplex Reagent Kit User Manual PART 2 Doc. No. 320293

Multiplex Fluorescent v2 RNAscope® Hydrogen Peroxide, Protease III RNAscope® Multiplex Fluorescent v2 Assay for Cultured Adherent Cells in 96-well Plate Format SOP 45-009A

Assay Type Sample Type RNAscope®/BaseScope™ Detection Assay

RNAscope®/BaseScope™ Detection Reagent User Manual

Leica BOND RX and Leica BOND RXm

FFPE Formalin-Fixed Paraffin‑Embedded

For 2.5 LS RED: RNAscope® 2.5 LS Reagent Kit – RED (BDZ9) User Manual Doc. No. UM-322150 RNAscope® 2.5 LS Reagent Kit – RED (BDZ11) User Manual Doc. No. 322150-USM

For 2.5 LS Duplex: RNAscope® 2.5 LS Duplex Reagent Kit (BDZ11) User Manual Doc. No. 322440-USM

RNAscope® 2.5 LSx Reagent Kit – BROWN and – RED For 2.5 LS BROWN: RNAscope® 2.5 LSx Reagent Kit – BROWN (BDZ15/BXD15) User Manual Doc. No. UM-322700

For 2.5 LS RED: RNAscope® 2.5 LSx Reagent Kit – RED (BDZ15/BXD15) User Manual Doc. No. 322750-USM

BaseScope™ LS Reagent Kit – RED BaseScope™ LS Detection Reagent Kit (BDZ11 or higher) User Manual Doc. No. 323600-USM-ULT

Ventana DISCOVERY™ ULTRA System

FFPE Formalin-Fixed Paraffin‑Embedded

RNAscope® VS Universal HRP Assay (BROWN) RNAscope® VS Universal HRP Assay (BROWN) User Manual Doc. No. 323200-USM-ULT

RNAscope® VS Universal AP Assay (RED) RNAscope® VS Universal AP Assay (RED) User Manual Doc. No. 323250-USM-ULT

RNAscope® VS Duplex Assay (RED/BROWN) RNAscope® VS Duplex Assay (RED/BROWN) User Manual Doc. No. 323300-USM-ULT

BaseScope™ VS Assay (RED), BaseScope™ VS Assay (RED) User Manual Doc. No. 323700-USM-ULT

Ventana DISCOVERY XT™ System*

FFPE Formalin-Fixed Paraffin‑Embedded

RNAscope® 2.5 VS Assay (BROWN) RNAscope® 2.5 VS Assay (BROWN) User Manual Doc. No. 322200-USM-XT

RNAscope® 2.5 VS Assay (RED) RNAscope® 2.5 VS Assay (RED) User Manual Doc. No. 322250-USM-XT

12 RNAscope® Getting Started Brochure

Get started today at acdbio.com/go

7707 Gateway Blvd, Newark, CA, 94560 | 1‑510‑576‑8800 (Main) | 1‑877‑576‑3636 (Toll Free)

This document is provided for informational use only. For Research Use Only (RUO), not for diagnostic use. BaseScope™ is a trademark and RNAscope®, HybEZ, and EZ-Batch are registered trademarks of Advanced Cell Diagnostics, Inc. in the United States or other countries. All rights reserved. ©2018 Advanced Cell Diagnostics, Inc. Doc # MK 51-124/ RevB/ 10012018

Chromogenic Singleplex Brown Chromogenic Singleplex Red Duplex Fluorescent Multiplex RNAscope® Assay Manual assays

Automated on Leica BOND RX System

Automated on DISCOVERY ULTRA and DISCOVERY XT* automated tissue staining systems by Roche Tissue Diagnostics

BaseScope™ Assay Manual assays

Automated on Leica BOND RX System

Automated on DISCOVERY ULTRA and DISCOVERY XT* automated tissue staining systems by Roche Tissue Diagnostics

Pharma Assay Services

Manual assays

Automated on Leica BOND RX System

Automated on DISCOVERY ULTRA and DISCOVERY XT* automated tissue staining systems by Roche Tissue Diagnostics

* Please note that the RNAscope® VS universal assays (Red, Brown or duplex) and the BaseScope™ assay are not available on the DISCOVERY XT

Product overview

Getting started with RNAscope

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