BaseScope Duplex Assay

CRISPR-mediated nucleotide deletion and BaseScope™ probe design. (A) CRISPR/Cas9-mediated gene editing was used to delete 49 nts from a wild-type (WT) gene, resulting in a novel junction sequence labeled “Edited”. (B) BaseScope™ paired oligo (1 ZZ) probes were designed to target either the 49 nts in the WT sequence or the Edited novel junction sequence.

Discern monoallelic or biallelic gene-editing status of cells in CRISPR/Cas9-treated liver with the BaseScope™ Duplex Assay. Detection of WT (closed triangle) and bi-allelic (open triangle) and mono-allelic (arrow) mutations in hepatocytes using BaseScope™ Duplex probes for the WT (green) or Edited (red) sequences.

Discern cell type-specific gene editing with the BaseScope™ Duplex Assay. CRISPR-mediated gene editing was detected in hepatocytes but not in the bile duct (BD) or endothelial cells (ECs) of the portal vein (PV) using BaseScope™ Duplex probes for the WT (green) or Edited (red) sequences.

Discern cell type-specific gene editing with the BaseScope™ Duplex Assay. CRISPR-mediated gene editing was detected in hepatocytes but not in the central vein (CV) using BaseScope™ Duplex probes for the WT (green) or Edited (red) sequences.

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:

Discerning bi-allelic vs. mono-allelic CRISPR-mediated mutations
Co-detection of circRNAs and linear RNAs
Simultaneous visualization of 2 splice variants or short targets
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.

Precise Visualization of CRISPR-Based Editing with Powerful RNAscope™ ISH Assays

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Visualization of in vivo gene editing heterogeneity at the transcript level with the BaseScope Duplex RNA in situ hybridization assay

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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

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

The BaseScope Duplex Manual Assay Workflow Requires:

BaseScope Duplex Catalog or Made-to-Order C1 & C2 Probes
BaseScope Duplex Positive Control Probes
BaseScope Duplex Negative Control Probes
RNAscope Control Slides (optional)
BaseScope Duplex Reagent Kit (Cat. no 323800) (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™ II Hybridization System
Brightfield microscope

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BaseScope™ Duplex Assay