Visualize a specific RNA locus at single cell sensitivity with spatial and morphological context

The BaseScopeTM Assay is a unique product from ACD, based on the same platform of proven and established RNAscope® technology that has been cited in over 900 publications in just 5 years. This powerful assay provides new data dimensions and unique insight into biological mechanisms. The BaseScopeTM Assay enables applications such as the detection of exon junctions/splice variants, short/highly homologous RNA sequences (50-300 bases), and point mutations at single cell sensitivity.

(1)Detecting biological events in cells and in situ using a single Z pair
(2)Highly specific and sensitive detection of RNA targets with down to ONE nucleotide differences
(3)Analysis of broad sample types: FFPE cells & tissues (TMAs), Fresh or fixed frozen tissues, Cultured cells

Exon Junctions / Splice Variants	Short Sequences	Point Mutations
	Gene therapy/AAV
Exon Junctions / splice variants	Short / highly homologous gene sequences (50-300nt)	Point Mutations (ACD validated point mutations only)
Circular RNA (circRNA)	T cell receptors (TCRs) and CDR sequences in T-cell clones	Short InDel
Gene fusion	Gene editing / CRISPR	Homologous
Gene knockout (KO)	Pre-miRNA
	Microexons
Splice Variant Example: Detection of EGFRvIII+ in glioblastoma with the BaseScope v2 Assay	Gene Editing Example: Discern cell type-specific gene editing with the BaseScope Duplex Assay	Point Mutation Example: Detection of KRAS G12D in KRAS mutation cell line with the BaseScope v2 Assay

Universal Pretreatment

Pretreatment reagents are used for blocking endogenous peroxidase activity and to optimally permeabilize properly prepared samples. These reagents have been optimized to better access target RNA and reduce optimization time in multiple tissues such as formalin-fixed, paraffin-embedded (FFPE) (including archival) tissue, fresh‑frozen (FF) tissue, fixed‑frozen tissue, tissue microarray (TMA), and cell preparations.

The pretreatment reagents listed in this section are for the manual RNAscope® and BaseScope™ Assays. Please refer to the appropriate sample preparation guides as shown in the User Manual Selection Guide available here to ensure your samples are prepared correctly for the RNAscope® and BaseScope™ detection assay.

RNAscope® 2.5 Universal Pretreatment Reagents (322380): this kit includes all the pretreatment reagents that may be used across different tissue types:

- RNAscope^® Target Retrieval Reagent
- RNAscope^® Hydrogen Peroxide Reagent
- RNAscope^® Protease III Reagent*
- RNAscope^® Protease IV Reagent*
- RNAscope^® Protease Plus Reagent*

Select the pretreatment reagents you need based on the sample type and detection assay type:

Tissue Type	Detection Assay Type	Reagent to use for pretreatment
FFPE (Formalin-Fixed, Paraffin-Embedded )	RNAscope® 2.5 HD Brown, Red, Duplex	RNAscope® Hydrogen Peroxide   RNAscope® Target Retrieval   RNAscope® Protease Plus*
	RNAscope® Multiplex Fluorescent v2	RNAscope® Hydrogen Peroxide   RNAscope® Target Retrieval   RNAscope® Protease III
	BaseScope™ Red	RNAscope® Hydrogen Peroxide   RNAscope® Target Retrieval   RNAscope® Protease III
Fixed Frozen	RNAscope® 2.5 HD Brown, Red, Duplex	RNAscope® Hydrogen Peroxide   RNAscope® Target Retrieval   RNAscope® Protease Plus
	RNAscope® Fluorescent Multiplex	RNAscope® Target Retrieval   RNAscope® Protease III
	RNAscope® Multiplex Fluorescent v2	RNAscope® Hydrogen Peroxide   RNAscope® Target Retrieval   RNAscope® Protease III
Fresh frozen	RNAscope® 2.5 HD Brown, Red, Duplex	RNAscope® Hydrogen Peroxide   RNAscope® Protease IV*
	RNAscope® Fluorescent Multiplex	RNAscope® Protease IV
	RNAscope® Multiplex Fluorescent v2	RNAscope® Hydrogen Peroxide   RNAscope® Protease IV
	BaseScope™ Red	RNAscope® Hydrogen Peroxide   RNAscope® Protease IV
Cultured Adherent cells	RNAscope® 2.5 HD Brown, Red, Duplex	RNAscope® Hydrogen Peroxide   RNAscope® Protease III
	RNAscope® Fluorescent Multiplex	RNAscope® Protease III
PBMC (Peripheral Blood Mononuclear Cells)/Non-Adherent Cells	RNAscope® Fluorescent Multiplex	RNAscope® Protease III

BaseScope™ RED Assay

The BaseScope™ Reagent Kit v2-RED, 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 a multitude of sample types mounted on slides. The BaseScope™ Assay is used for the detection of short RNA targets (50-300 bases), highly homologous sequences, RNA mutations, and exon junctions for the detection of splice variants, circular RNA, and gene fusion. The BaseScope™ Assays do not require an RNase-free environment that is used for traditional RNA ISH.

Applications of the BaseScope™ RED Assay

Exon Junctions/ Splice Variants	Short/Homologous Target Sequences	Point Mutations*
Exon junctions/splice variants	Short targets/highly homologous gene sequences (50-300nt)	Point mutation
Circular RNA (cirRNA)	T cell receptors (TCRs) and CDR sequences in T-cell clones	Short InDel
Gene fusion	pre-miRNA	Homologues
Gene knockout (KO)	Gene editing/CRISPR	* ACD Validated Point Mutations Only

BaseScope™ Duplex Assay

The BaseScope™ Duplex Assay is based on ACD’s patented signal amplification and background suppression technology. The BaseScope™ assay leverages the same platform of the proven and established RNAscope® technology but expands the detection capabilities, enabling the detection of short RNA target sequences and exon junctions in cells and tissues with morphological context, thereby providing new data dimensions and unique insight into biological mechanisms.The BaseScope™ Duplex Assay can be used for simultaneous visualization of two RNA targets while maintaining single cell resolution.

Applications of the BaseScope™ Duplex Assay include:

1. Discerning bi-allelic vs. mono-allelic CRISPR-mediated mutations
2. Co-detection of circRNAs and linear RNAs
3. Simultaneous visualization of 2 splice variants or short targets
4. Profiling expression of a splice variant, short target, or gene edit in a cell-specific manner

One major application for the BaseScope™ Duplex Assay is to identify gene edits in specific cell populations by duplexing RNA ISH cell marker probes and edit-specific probes. The BaseScope™ Duplex assay can discern if the CRISPR/Cas9-mediated mutation is mono- or bi-allelic with duplex of WT and edited probes.

BaseScope™ Duplex Reagent Kit provides reliable results which are ideal for archiving due to the permanent staining visible under any standard bright field microscope. The BaseScope™ Duplex Reagent Kits are designed to provide more intense staining when low copy target gene expression is anticipated (1–20 copies per cell). The BaseScope™ Duplex reagent kits are compatible with BaseScope™ probes.

Basescope Duplex Assay Intro Packs

For customers using BaseScope™ Duplex 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.

1. BaseScope Duplex Reagent Kit Intro Pack- Hs (cat. no 323870)
2. BaseScope Duplex Reagent Kit Intro Pack- Mm (cat. no 323871)