RNAscope^® HiPlex Assays

Introducing RNAscope^® HiPlex assay to expand your in situ hybridization potential. Now allowing simultaneously detection of up to 12 different RNA targets per slide mounted sample. Higher plexing of up to 12 different RNA targets is enabled by employing cleavable fluorophores and iterative detection with the RNAscope^® HiPlex 12 Reagents Kit.

“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

Clear selection

 

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

 

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Technology Overview:

RNAscope^® HiPlex assay requires the reagent kit in conjunction with user specified target probes which contain a mixture of short oligonucleotides designed to bind to a specific target RNA. Each target probe must be assigned to a different probe tail/type (T1-T12). After a series of highly effective and specific signal amplifications, the signal is detected iteratively, where a single RNA transcripts for up to four target genes at a time is visualized as punctate dots in four distinct fluorescent channels. The assay uniquely employs the cleavable versions of fluorophores AF488, Atto550, Atto647 and AF750, the iterative process is to be repeated until all 12 targets are detected. The fluorophore cleaving procedure is rapid with minimal/no effect on RNA and tissue morphology and is independent from a probe stripping protocol. Images from various rounds can be merged together using RNAscope^® HiPlex Image Registration Software.

Getting started

The RNAscope^® HiPlex Assay8 Workflow Requires the following:

• RNAscope^® HiPlex Target Probes (Catalog or Made-to-Order T1 to T12 Probes,  not compatible with the RNAscope^® probes with C1-C4 designation)
• RNAscope^® HiPlex Positive Control Probes (species specific)
• RNAscope^® HiPlex Negative Control Probes (Cat. No. 324341)
• RNAscope^® HiPlex Probe Diluent (Cat. No. 324301)
• RNAscope^® RNAscope^® HiPlex12 Reagents Kit (488, 550, 647, 750) (Cat. No. 324107) (Includes Pretreatment Kit, detection kit, cleaving reagent and wash buffer)
• RNAscope^® HiPlex12 Reagents Kit (488, 550, 647) (Cat. No. 324108) (Includes Pretreatment Kit, detection kit, cleaving reagent and wash buffer)
• ImmEdge Hydrophobic Barrier Pen (Cat. No. 310018)
• HybEZ™ Hybridization System
• Fluorescent microscope with detection capability to detect DAPI, AF488, Atto550, Atto647N and AF750

If using RNAscope^® HiPlex Assay for the first time, our Intro Packs include species-specific positive control probe, universal negative control probe, probe diluent, the Reagent Kit, the 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.

RNAscope^® Intro Pack for HiPlex12 Reagents Kit (488, 550, 647) - Fresh/Fixed Frozen – Mm (Cat No. 324194)
RNAscope^® Intro Pack for HiPlex12 Reagents Kit (488, 550, 647) - Fresh/Fixed Frozen – Rn (Cat No. 324109)
RNAscope^® Intro Pack for HiPlex12 Reagent Kit - Fresh/Fixed Frozen – Mm (Cat. No. 324102)
RNAscope^® Intro Pack for HiPlex12 Reagent Kit - Fresh/Fixed Frozen – Rn (Cat. No. 324104)

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.