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RNAscope™ HiPlex v2 Assays

Detecting Drd1a and Drd2 major and minor populations using RNAscope HiPlex assay on same sagittal section of mouse brain

Detecting Drd1a and Drd2 major and minor populations using RNAscope HiPlex assay on same sagittal section of mouse brain

Visualizing striatal Drd1a and Drd2 Medium Spiny Neurons (MSNs) using the RNAscope HiPlex assay for 12 targets in fresh frozen mouse brain sections targets are Drd1a, Drd2, Foxp1, Pcdh8, Synpr (in white), Htr7, Meis2, Calb1, Crym, Cnr1, Wfs1, Th (in yellow)

Visualizing striatal Drd1a and Drd2 Medium Spiny Neurons (MSNs) using the RNAscope HiPlex assay for 12 targets in fresh frozen mouse brain sections targets are Drd1a, Drd2, Foxp1, Pcdh8, Synpr (in white), Htr7, Meis2, Calb1, Crym, Cnr1, Wfs1, Th (in yellow)

Profiling different cell types within the TME. 12 target specific marker probes were used to detect immune cells, tumor cells, chemokines and cytokines in Lung cancer

Profiling different cell types within the TME. 12 target specific marker probes were used to detect immune cells, tumor cells, chemokines and cytokines in Lung cancer

Positive control probe analysis in stomach cancer tissue with RNAscope HiPlex 12-plex positive control probe

Positive control probe analysis in stomach cancer tissue with RNAscope HiPlex 12-plex positive control probe

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

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

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 expression data, enabling comprehensive spatial studies from 12 targets in FFPE samples and up to 48 targets in Fresh and Fixed Frozen sample types.

We love the RNAscope HiPlex assay!  RNAscope was already an integral part to our workflows to evaluate and validate the results we get from single cell sequencing experiments.  The capabilities of the HiPlex assay allow us to visualize several different cell types and/or states in the same slice of tissue which provides critical information on spatial distribution and relationships.  The protocol worked exactly as advertised and very similar to current multiplex RNAscope.  Additionally, the free ACD image registration software worked like a charm and was very straightforward to align all of the images from multiple rounds of imaging.”
-Samuel Marsh, PhD and Connor Dufort,
Lab of Dr. Beth Stevens, Boston Children’s Hospital

Drd1a
Synpr
Crym
Drd2
Htr7
Cnr1
Foxp1
Meis
Wfs1
Pcdh8
Calb1
TH
 

Visualizing striatal Drd1a and Drd2 Medium Spiny Neurons (MSNs) using the RNAscope HiPlex assay for 12 targets in fresh frozen mouse brain sections targets are Drd1a, Drd2, Foxp1, Pcdh8, Synpr, Htr7, Meis, Calb1, Crym, Cnr1, Wfs1, TH

One major application for the RNAscope HiPlex Assay is to validate gene signatures with spatial context at the single cell level.

Hiplex Probe Sets

Incorporating RNAscope HiPlex Assay into single cell RNA sequencing workflows

App Note

Spatial Profiling of Immune Cell Gene Signatures in the Tumor Microenvironment

Brochure

Enhancing your Spatial Genomics story

Mapping Single Cells Back to the Tissue

 

Technology Overview:

The RNAscope HiPlex v2 assay is capable of multiplex fluorescent detection of up to 12 targets in fresh/fixed frozen and FFPE tissues. In this assay, after a series of highly effective and specific signal amplifications, the signal is detected iteratively, where up to four target genes are visualized in four distinct fluorescent channels. Similar to previous RNAscope assays, a single RNA transcript can be visualized as a punctate dot. The assay uniquely employs the cleavable versions of different fluorophores, and the iterative process is to be repeated until all 12 targets are detected. The fluorophore cleaving procedure is rapid with minimal to no effect on RNA and tissue morphology since it does not strip away the bound probe. Finally, the images from various rounds can be merged using RNAscope HiPlex Image Registration Software v2. This software allows the user to toggle and overlay expression of only select targets thereby making data interpretation very simple.

 

Leveraging our HiPlex assay chemistry, this new RNAscope HiPlexUp reagent enables expansion of  our multiplexing capability of the RNAscope HiPlex assay to 48-targets.

The workflow contains a series of highly effective and specific signal amplifications, whereby running 4 cycles of 3 rounds each, allows for visualization of 4 targets by iterative detection after each round.

Ordering Information

RNAscope HiPlexUp Reagent            Cat No. 324190

 

Getting started

If using RNAscope HiPlex Assay for the first time, start with our Intro Packs.

Intro-packs include everything you need to run the assay - Reagent Kit, Species-Specific Positive Control Probe, Universal Negative Control probe, Probe Diluent, ImmEdge hydrophobic barrier pen and the RNAscope HiPlex Image Registration Software.

Be sure to order your specific target probes with the specific tails/type ranging from T1 to T12 separately.

 

Product NameProduct Code
RNAscope Intro Pack for HiPlex12 Reagents Kit (488, 550,650,750) - Mouse324440

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

324441
RNAscope Intro Pack for HiPlex12 Reagents Kit (488, 550,650,750) - Human324442
RNAscope Intro Pack for HiPlex12 Reagents Kit ((488, 550,650) - Mouse324443
RNAscope Intro Pack for HiPlex12 Reagents Kit (488, 550,650) - Rat324444
RNAscope Intro Pack for HiPlex12 Reagents Kit (488, 550,650) - Human324445

 

The RNAscope HiPlex Assay Workflow Requires the following:

 

Assay ComponentProduct NameProduct Code

Reagent Kit


RNAscope HiPlex12 Reagents Kit (488, 550, 650, 750) v2

RNAscope HiPlex12 Reagents Kit (488, 550, 650) v2


324409

324419

Species Specific Positive Control Probe



RNAscope HiPlex12 Positive Control Probe – Rn v2

RNAscope HiPlex12 Positive Control Probe – Hs

RNAscope HiPlex12 Positive Control Probe – Mm v2



324434

324311

324433

Universal Negative Probe

RNAscope HiPlex12 Negative Control Probe

324341

Probe Diluent

RNAscope HiPlex Probe Diluent 

324301

Software

RNAscope HiPlex Image Registration Software

300065

Hydrophobic Barrier Pen

Hydrophobic Barrier Pen

310018

Control Slide

RNAscope Control Slide -Mouse 3T3 Cell Pellet

310023

Hybridization System

HybEZ™ Hybridization System

 

Microscope Detection Capabilities

Fluorescent microscope with detection capability to detect DAPI, AF488, Atto550, Atto647N and AF750

 

 

 

Guidelines for Fluorophore Assignment for RNAscope HiPlex Assay

The RNAscope HiPlex Assay amplification system is designed to maintain a high signal to noise ratio, however, fluorophore brightness is an important consideration when building your panel. Please see the table below for our recommendations for each fluorophore.

Microscopy ChannelProsConsRecommendations
Fluorescein - Green
For Alexa Fluor 488
Visible with naked eyeLeast distinct from most tissue autofluorescenceHigh Expressor

Cyanine 3 - Orange

For Dylight 550

Visible with naked eyeNoneLow Expressor or Expression Level Unknown
Cyanine 5 - Far Red

For Dylight 650

Easily differentiated from autofluorescenceNot visible to the naked eyeLow Expressor or Expression Level Unknown

Cyanine 7 - Far Red

For Alex Fluor 750

Easily differentiated from autofluorescenceNot visible to the naked eyeLow Expressor
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