RNAscope™ HiPlex v2 Assays
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
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.
Incorporating RNAscope HiPlex Assay into single cell RNA sequencing workflows
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 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, 550,650) - Human | 324445 |
The RNAscope HiPlex Assay Workflow Requires the following:
| Assay Component | Product Name | Product Code | ||||||||
|---|---|---|---|---|---|---|---|---|---|---|
Reagent Kit |
|
| ||||||||
Species Specific Positive Control Probe |
|
| ||||||||
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 Channel | Pros | Cons | Recommendations |
|---|---|---|---|
| Fluorescein - Green For Alexa Fluor 488 | Visible with naked eye | Least distinct from most tissue autofluorescence | High Expressor |
Cyanine 3 - Orange For Dylight 550 | Visible with naked eye | None | Low Expressor or Expression Level Unknown |
| Cyanine 5 - Far Red For Dylight 650 | Easily differentiated from autofluorescence | Not visible to the naked eye | Low Expressor or Expression Level Unknown |
Cyanine 7 - Far Red For Alex Fluor 750 | Easily differentiated from autofluorescence | Not visible to the naked eye | Low Expressor |
