RNAscopeTM HiPlex v2 ASSAY ENHANCING YOUR SPATIAL GENOMICS STORY

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Spatial transcriptomics enabled by direct visualization of RNA molecules in intact samples holds great promise in resolving heterogenous tissues at single cell and sub-cellular resolution. Identifying genes that are differentially expressed in distinct cell populations provide insights into cellular organization and function of diverse cell types in healthy and disease states.

FIGURE 1. RNAscope HiPlex assay on human FFPE cervical cancer tissue, using Alexa 488, DL550 and DL650 fluorophores for the detection of HiPlex12 positive control probes RPL5, B2M, and ACTB.

RPL5

B2M

ACTB

RPL5 B2M ACTB

INTRODUCING HiPlex ASSAY RNAscope HiPlex v2 assay is an expansion of the RNAscope HiPlex assay platform, an advanced in situ hybridization (ISH) tool to understand vital gene expression patterns at single cell resolution.

The new assay generates precise gene expresssion data, allowing researchers to conduct comprehensive spatial studies from 12 targets in FFPE samples and up to 48 targets in Fresh and Fixed Frozen sample types.

The assay enables direct visualization of the transcript by eliminating the need for difficult bioinformatics methods to analyze and interpret scRNA-seq data. It also provides accurate and reliable data with a simple workflow, making it the assay of choice for translational research and biomarker discovery.

This highly sensitive and multi-plexing assay can be performed on commonly available laboratory equipment without the need to invest in expensive capital equipment.

ASSAY FEATURES

• Higher Plexing Capability

• All Tissue Types - FFPE, Fresh and Fixed Frozen

• Single Molecule Detection Capability

• True Single Cell Resolution

• No Degradation Bias

• Enzyme Free Signal Amplification

• Effortless Probe Design

• Novel Image Registration Software

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ENABLING MULTITUDE OF APPLICATIONS RNAscope HiPlex assay has applications across multiple research areas including novel applications such as complex tissue profiling and single cell validation.

COMPLEX TISSUE PROFILING

Complex tissues are comprised of multiple cell types with dynamic interactions that are governed by their spatial organization. The RNAscope HiPlex assay can help establish gene signatures within tissues and uncover critical pathways implicated in disease pathology.

Heterogenous Tissue Select Target Genes Based on Research Interest

Spatial Mapping of Targets Using RNAscope HiPlex Assay

Map Signaling Pathways

Profile the Tumor

Microenvironment

Study Pathogen- Induced Immune

Response

Compare Expression

of Target Genes Between Disease

and Normal Tissues

B Cells

CD8+ T cells

CD8+ T cells

γδ T cells

γδ T cells

Mast Cells

Carcinoma-associated Fibroblasts

Carcinoma-associated Fibroblasts

N2 Neutrophils

Th1 Cells

Th1 Cells

Th1 Cells

Regulatory B Cells

Type II NKT cells

Type I NKT cells

IFN-γ TNF-α

IFN-γ

IFN-γ TNF-α

Type I NKT cells

M2 Macrophages

M1 Macrophages

M1 Macrophages

M2 Macrophages

CCL2

N1 Neutrophils

NK Cells

CD8+ T cells

IFN-γ TNF-α

IFN-γ IL-2

TNF-α

IFN-γ IL-2 TNF-α

IFN-γ IL-12

TNF-α NO IFN-γ

TNF-α

IL-4 IL-13

CCL2 IL-10 TGF-β

IL-4 IL-5 IL-9 IL-10 IL-13

IL-10 TGF-β IL-35

IL-10 TGF-β IL-35

IL-10 TGF-β

IL-10, TGF-β Arginase 1

IFN-γ IL-10 TGF-β

IL-35

Monocytes

Exhausted NK Cells Exhausted

CD8+ T Cells

Th2 Cells

IFN-g TNF-α

NK Cells

Monocytes

MDSCs

DC

Tregs

MDSCs

TregsNK Cells

FIGURE 3. Illustration of complex tissue profiling workflow using RNAscope HiPlex assay to resolve cellular heterogeneity by spatially validating targets of interest within intact tissue samples across various research areas.

ASSAY WORKFLOW The new HiPlex assay offers a powerful and efficient plexing tool for validation and visualization of multiple targets with a vast catalog of RNAscope probes and rapid turn-around-time for custom probes for any target. The advanced capabilities of this assay increases confidence in higher plexing data platforms that traditionally rely on grind and bind or enzymatic amplification based approaches.

ZZ ZZZZ ZZ Target 1 Target 12

Target Probe Hybridization

ZZ ZZ ZZ ZZ Target 1 Target 12

Signal Amplification

ZZ ZZ ZZ ZZ Target 1 Target 4

Label Probe Hybridization

Image Registration

Imaging

Repeat Label Probe Hybridization

ZZ ZZ ZZ ZZ Target 1 Target 4

ZZ ZZ ZZ ZZ Target 5 Target 8

Fluorophore Cleavage

ZZ ZZ ZZ ZZ Target 1 Target 4

3-4 Cycles

FIGURE 2. RNAscope HiPlex assay provides signal amplification for simultaneous visualization of up to 12 RNA targets in FFPE and up to 48 targets in fresh and fixed frozen samples by performing iterative fluorescent imaging rounds.

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SINGLE CELL TRANSCRIPTOMIC VALIDATION

The RNAscope HiPlex assay provides a multiplexing solution for orthogonal confirmation and spatial mapping of scRNA-seq data by enabling simultaneous visualization of up to 12 target genes.

Bulk Tissue Single-Cell

Dissociation Single-Cell RNA-Seq

Validate & Spatially Map

FIGURE 5. Incorporation of spatial validation into single cell sequencing based transcriptomic workflows with the RNAscope technology.

FIGURE 4. Detecting immune cells, chemokines, cytokines and cancer cell markers by spatially profiling the cervical cancer TME.

CCL22 CXCL10 IFNG VEGF PD1 ARG1 HIF1A CD3 PDL1 CD68 CD163 CD8

Drd2 Striatal MSNs

FIGURE 6. Simultaneous visualization and characterization of the D1 and D2 subtypes in the mouse striatum with the RNAscope HiPlex assay. The D1 subtypes are characterzied by Drd1a, Foxp1, Pcdh8, Meis2, Dner, Cnr1, Crym, and Wfs1 (A), whereas the D2 subtypes are characterized by Drd2, Calb1, Cartpt, Synpr, Htr7, Crym, Cnr1, and Th. (B) Insets show cells expressing markers indicative of the major D1 (A) and D2 (B) subtypes. Validation of striatal neuronal subpopulations identified by scRNAseq as described in Gokce et al., Cell Rep, 2016.

RNAscope HiPlex imaging of Drd1a and Drd2 medium spiny neuronal (MSN) subtypes

Drd1a Striatal MSNs

A

B

Drd1a Drd2 Foxp1 Pcdh8 Synpr Htr7 Meis2 Calb1 Crym Cnr1 Wfs1 Th

Drd1a Foxp1 Pcdh8 Meis2 Cnr1 Wfs1 Th

Drd2 Synpr Htr7 Calb1 Crym Th

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PRODUCT INFORMATION AND RESOURCES

ADDITIONAL RESOURCES • HiPlex webpage – acdbio.com/rnascope-hiplex-assay

• Single cell webpage - acdbio.com/science/applications/research-solutions/single-cell-analysis

• Webinars - acdbio.com/technical-support/learn-more/recorded-webinars

• Tech support resources - acdbio.com/technical-support/support-overview

Research areas enabled by RNAscope HiPlex applications -

Immunology/ Inflammation

Neuroscience Immuno-oncologyOncology Translational Research

Stem Cell Research

Leveraging our HiPlex assay chemistry, RNAscope HiPlexUP reagent enables expansion of our multiplexing capability of the RNAscope HiPlex assay to 48 targets on Fresh and Fixed Frozen sample types.

Intro-packs include all the components needed to run the assay - HiPlex Reagent Kit, HiPlex Positive and Negative Control Probes, HiPlex Probe Diluent, HiPlex Control Slides, HiPlex Image Registration Software and Hydrophobic Barrier Pen

PRODUCT NAME PRODUCT

CODE

RNAscope HiPlexUp Reagent 324190

ASSAY CHECKLIST

HiPlex Reagents Kit Pretreatment Kit, Detection Kit, and Wash Buffer and Cleaving Reagent Kits

HiPlex Control Probes Positive and Negative Control probes

HiPlex Probe Diluent Probe Preparation Reagents

RNAscope HiPlex Image Registration Software Image Registration Software to register and merge the images acquired after performing the HiPlex Assay

Hydrophobic Barrier Pen Heat-stable water-repellent barrier to keeps reagents localized on tissue sections

HiPlex Control Slides Recommended set of control slides

START WITH INTRO-PACKS

PRODUCT NAME PRODUCT

CODE

RNAscope Intro Pack for HiPlex12 Reagents Kit (488, 550, 650, 750) - Mouse 324440

RNAscope Intro Pack for HiPlex12 Reagents Kit (488, 550, 650, 750) - Rat 324441

RNAscope Intro Pack for HiPlex12 Reagents Kit (488, 550, 650, 750) – Human 324442

RNAscope Intro Pack for HiPlex12 Reagents Kit (488, 550, 650) - Mouse 324443

RNAscope Intro Pack for HiPlex12 Reagents Kit (488, 550, 650) - Rat 324444

RNAscope Intro Pack for HiPlex12 Reagents Kit (488, 550, 65) - Human 324445

https://acdbio.com/rnascope-hiplex-assays https://acdbio.com/science/applications/research-solutions/single-cell-analysis https://acdbio.com/technical-support/learn-more/recorded-webinars https://acdbio.com/technical-support/support-overview

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NOTES

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NOTES

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BR_HiPlex Brochure_STRY0144164MK 51-167 HiPlex v2 Assay Brochure

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