RNAscope Fluorescent Multiplex Assays

Spatial analysis of gene expression is an essential tool for comprehensive studies of complex, highly heterogeneous tissues, such as the brain, tumors, and developing organs. Traditional RNA-ISH can be challenging due to low sensitivity, inconsistent detection accompanied with limited plexing. RNAscope® Fluorescent Multiplex assays provide exceptional sensitivity, allowing single-molecule detection of RNA targets at the single cell level. The RNAscope® Multiplex Fluorescent assays are ideal for simultaneous detection studies of any genes in nearly any tissue-type using fluorescent labels. We offer with various levels of plexing to:

(1)FIND cells of interest by using cell specific markers
(2)CONFIRM cells by simultaneous detection of additional targets
(3)CHARACTERIZE cellular heterogeneity of complex tissues by mapping and identifying multiple cell subtypes simultaneously

“Adding a histological context to scRNAseq data is important for fully understanding the biology of distinct cell populations, and HiPlex RNAscope is a great way to do this. The ability to stain a single section of tissue with up to 12 mRNA probes makes HiPlex RNAscope a powerful tool for distinguishing cells types in situ. While the HiPlex RNAscope protocol is relatively straightforward to conduct, ACD Bio has always been there to help when I need sales or technical assistance. I would recommend HiPlex RNAscope to anyone looking to visualize distinct populations of cells from scRNAseq data in situ.”
                                                                                                                    ---Jocob Swanson, Hospital for special surgery, NY

Which RNAscope® Multiplex kit is best for me - three kits are available

	RNAscope® HiPlex	RNAscope® Multiplex Fluorescent v2	RNAscope® Fluorescent Multiplex
Plexing offered for target detection	12 Plex capability enabled with RNAscope® 12Plex Ancillary Kit (Cat. No. 324140)*	4 Plex capability enabled with RNAscope® 4-Plex Ancillary Kit for Multiplex Fluorescent v2 (Cat. No. 323120)*	3 Plex capability
Compatible sample preparation	Fresh frozen, Fixed frozen	FFPE, fixed frozen (low expressors)** Cells and fresh frozen (if highly autofluorescent)	Fresh frozen, Cells, Fixed frozen (high expressors)
Additional items required	None	Opal dyes must be purchased from Akoya Biosciences.	None
Workflow length	9 hr (Recommended to split over 3 days)	14 hr (Recommended to split over 2 days)	6-8 hours
Fluorophores used/recommended	Alexa Fluor-488, Atto-550, Atto-647N, Alexa Fluor-750 fluorophores provided in the detection kit (Fluoro reagents)	Purchased from Akoya Biosciences:  3-plex:  Opal™ 520 Reagent Pack (Cat No: FP1487001KT)  Opal™ 570 Reagent Pack (Cat No: FP1488001KT)  Opal™ 690 Reagent Pack (Cat No: FP1497001KT)    4-plex:  Opal™ 520 Reagent Pack (Cat No: FP1487001KT)  Opal™ 570 Reagent Pack (Cat No: FP1488001KT)  Opal™ 620 Reagent Pack (Cat No: FP1495001KT)  Opal™ 690 Reagent Pack (Cat No: FP1497001KT)	Alexa Fluor-488, Atto-550 and Atto-647N fluorophores provided in the detection kit (Amp 4)
Cleavable Fluorophore	Yes	No	No
ISH-IF compatibility	Compatible	Compatible: please refer to Doc# 323100-TN	Not recommended
Recommended for	Low autofluorescing tissues	High autofluorescing tissues	Low autofluorescing tissues
Probes Designation	RNAscope® HiPlex Probes: T1, T2, T3, T4, ……T12	RNAscope® Probes: C1, C2, C3, C4	RNAscope® Probes: C1, C2, C3
Recommended Filter sets on the Fluorescent microscope:	Ex Max 358 nm/Em Max 461 nm (DAPI)  Ex Max 494 nm/Em Max 517 nm (FITC)  Ex Max 550 nm/Em Max 570 nm (Cy3)  Ex Max 648 nm/Em Max 667 nm (Cy5)  Ex Max 753 nm/Em Max 777 nm (Cy7)	Ex Max 358 nm/Em Max 461 nm (DAPI)  Ex Max 494 nm/Em Max 525 nm (FITC)  Ex Max 550 nm/Em Max 570 nm (Cy3)  Ex Max 588nm/Em Max 616 nm (Tx Red)  Ex Max 676 nm/Em Max 694 nm (Cy5.5)	Ex Max 358 nm/Em Max 461 nm (DAPI)  Ex Max 494 nm/Em Max 517 nm (FITC)  Ex Max 550 nm/Em Max 570 nm (Cy3)  Ex Max 648 nm/Em Max 667 nm (Cy5)
Mounting medium	Prolong Gold	Prolong Gold	Prolong Gold
Cat No	324100	323100	320850
Description	RNAscope® Fluorescent Multiplex Reagent Kit designed using ACD's proprietary amplification strategy. The assay uses Alexa Fluor-488, Atto-550, Atto-647N and Alexa Fluor-750 for detecting up to 12 targets on the same tissue section.	TSA-based Fluorescent RNAscope®detection assay for up to 4-plex capability using Opal fluorophores (sold separately). Ideal for FFPE tissues	RNAscope® Fluorescent Multiplex Reagent Kit employs Alexa Fluor-488, Atto-550 and Atto-647N fluorophores for up to 3-plex detection capability.
Area of research	Neuroscience	Immuno-oncology (CD8, PD1, PDL1), Oncology, Neuroscience	Neuroscience

 

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.

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.

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

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

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 Atto 550	Visible with naked eye	None	Low Expressor or Expression Level Unknown
Cyanine 5 - Far Red  For Atto 647N	Easily differentiated from autofluorescence	Not visible to the naked eye	Low Expressor
Cyanine 7 - IR  For Alexa Fluor 750	Easily differentiated from autofluorescence	Not visible to the naked eye	Low Expressor

RNAScope® Multiplex Fluorescent V2 Assay

The RNAscope® Multiplex Fluorescent v2 assays provides exceptional sensitivity and allows single-molecule detection of up to four RNA targets simultaneously. The significant signal boost provided by simultaneous background noise suppression from patented signal amplification technology for ISH when combined with Akoya Biosciences proprietary Tyramide Signal Amplification technology (TSA™) for visualization allows expanded applications such as ISH on FFPE tissues and dual ISH-IHC.

Technology Overview:

RNAscope® Multiplex Fluorescent Assay v2 allows simultaneous detection of up to four RNA targets within a single sample using up to four target probes and the nuclei can be counter stained and visualized using DAPI fluorescent dye. The four target probes, each designed a different probe channel—C1, C2, C3, or C4 contain several components. The lower part of the double Z probe is designed to hybridize to a specific RNA target. The upper part of the double Z probe, along with the preamplifiers, amplifiers and dyes are assigned to a specific probe channel. For 4-plex assay we recommend using the Opal 520 (FITC range), Opal 570 (Cy 3 Range), Opal 620 (Texas Red Range) and Opal 690 (Cy 5.5 Range) dyes. The sequential assay workflow provides flexibility to visualize any probe in any of the TSA linked fluorophore channels.

Guidelines for Fluorophore Assignment for RNAscope® Multiplex Fluorescent v2 Assay

The RNAscope® Multiplex Fluorescent v2 Assay is flexible with fluorophore assignments and the end user can choose any fluorophore (from the recommended set) for visualizing any targets, however, fluorophore brightness is an important consideration when assigning them to each target. Please see our recommendations in the table below:

Microscopy Channel	Pros	Cons	Recommendations
Fluorescein - Green  For Opal™ 520	Visible with naked eye	Least distinct from most tissue autofluorescence	High Expressor
Cyanine 3 - Orange  For Opal™ 570	Visible with naked eye	None	Low Expressor or Expression Level Unknown
Texas Red - Red  For Opal™ 620	Easily differentiated from autofluorescence	None	Low Expressor or Expression Level Unknown
Cyanine 5.5 - Near IR  For Opal™ 690	Easily differentiated from autofluorescence	Not visible to the naked eye	Low Expressor

RNAscope® Fluorescent Multiplex Assay

RNAscope® Fluorescent Multiplex Assay is our first multiplex assay and is based on our patented signal amplification and background suppression technology. The assay offers a novel and proprietary method of in situ hybridization (ISH) to achieve single cell resolution for RNA detection in a multitude of slide mounted samples.

Technology Overview:

RNAscope® Fluorescent Multiplex assay allows simultaneous detection of 3 RNA targets within a single sample using 3 target probes, each optimized for a different probe channel—C1, C2 or C3. Components within each multiplex probe are unique. The lower part of the double Z probe is designed to hybridize to a specific RNA target. The upper part of the double Z probe, along with the preamplifiers, amplifiers and dyes are assigned to a specific probe channel. Signal detection is performed using dyes with excitation and emission properties equivalent to those of FITC , Cy3 and Cy5 dyes.

Guidelines for Fluorophore Assignment for RNAscope® Fluorescent Multiplex Assay

The RNAscope® Fluorescent Multiplex Assay offers three Alternate display modules and the end user can choose Alt A, B or C for visualizing the targets, however, fluorophore brightness is an important consideration when assigning them to each target. Please see our recommendations in the table below:

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 Atto 550	Visible with naked eye	None	Low Expressor or Expression Level Unknown
Cyanine 5 - Far Red  For Atto 647N	Easily differentiated from autofluorescence	Not visible to the naked eye	Low Expressor