RNAscope™ Plus smRNA-RNA Assay

Small regulatory RNAs, including miRNAs, are instrumental in regulating human health and have been associated with a wide range of different diseases. Oligonucleotide therapies, including antisense oligonucleotides (ASOs) and RNA interference (RNAi), represent a unique class of gene therapies that have the potential to provide highly focused, long-lasting therapeutic effects by utilizing similar cellular processes.

Advanced Cell Diagnostics miRNAscope ISH technology has become the gold standard for identifying the cellular localization of endogenous miRNAs and measuring delivery efficiency of therapeutic oligonucleotides in preclinical animal model systems. Combining proprietary RNAscope and miRNAscope ISH technologies, we are now introducing the RNAscope Plus smRNA-RNA assay which is a valuable new tool to quantify changes in gene expression and cellular function in response to the introduction of regulatory RNAs, essential for optimization of therapeutic efficacy and safety.

Key Features:

  • Unlock the potential to detection 1 small RNA along with up to 3 mRNA targets
  • Visualize antisense oligonucleotides (ASOs), microRNAs (miRNAs), small interfering RNAs (siRNAs), and other smaller RNAs that are 17-50 nt in length along with 3 RNA targets
  • Visualize gene regulation with morphological context
  • Validate miRNA biomarkers in intact tissues
  • Assess small RNA therapeutic delivery mechanism
  • Evaluate biodistribution and efficacy of therapy
  • Add visual dimension to heterogeneous tissue biology and analysis
  • Combined ACD patented signal amplification system and the Tyramide Signal Amplification (TSA™) technology


Watch this short video and learn more about RNAscope Plus:

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Introducing the RNAscope Plus small RNA-RNA Fluorescent Assay

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Biodistribution, safety and efficacy assessment of small RNAs with the new RNAscopeTM Plus small RNA-RNA co-detection Assay

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Technology Overview:

RNAscope Plus smRNA-RNA assay allows simultaneous detection of 1 smRNA and up to 3 RNA targets within a single sample using up to four target probes and the nuclei can be counterstained and visualized using DAPI fluorescent dye. The four target probes, each designed in a different probe channel—S1 for smRNA, and C2, C3, or C4 for RNA targets. The assay is compatible with formalin-fixed, paraffin-embedded (FFPE) and fresh frozen (FF) tissue. 

The RNAscope Plus smRNA-RNA Assay Workflow Requires the following:

  • Target Probes in channel S1 for smRNA and C2, C3, C4 for RNA
  • RNAscope™ Plus smRNA-RNA HD Reagents Kit (Includes RNAscope™ Plus smRNA-RNA HD Detection Kit, RNAscope Multiplex TSA Buffer, RNAscope™ Wash Buffer Reagents, RNAscope™ H2O2 and Protease Reagents) (Cat. No. 322785)
  • RNAscope™ Plus smRNA-RNA 4-plex Positive Control Probe-Hs (Cat. No. 323361) or
  • RNAscope™ Plus smRNA-RNA 4-plex Positive Control Probe-Mm (Cat. No. 323371) or
  • RNAscope™ Plus smRNA-RNA 4-plex Positive Control Probe-Rn (Cat. No. 323381) or
  • RNAscope™ Plus smRNA-RNA 4-plex  Negative Control Probe (Cat. No. 323391)
  • ImmEdge Hydrophobic Barrier Pen (Cat. No. 310018)
  • HybEZ™ Hybridization System
  • Opal™ Dyes from Akoya Biosciences or Vivid Dyes from ACD
  • Fluorescent microscope with capability to detect DAPI, Opal™ 520, Opal™ 570, Opal™ 690 and Opal™ 780

Guidelines for Microscope Channel Selection:

RNAscope Plus smRNA-RNA assay supports interrogation of up to 4 target probes. The assay uses four target probes with each probe designed in a different channel – the S1 channel for smRNA and C2, C3 and/or C4 channels for other RNA targets.  The nuclei can be counterstained and visualized using DAPI fluorescent dye. For viewing of 4 plex assay, a multiplexed biomarker imaging system is required, such as Nuance FX Multiplex Biomarker Imaging System, Vectra 3.0 Automated Quantitative Pathology Imaging System or Mantra Quantitative Pathology Workstation from Perkin Elmer.

Fluorophore options depend on the imaging systems available. Two color combination examples are shown in the following table. Multispectral un-mixing capability are required for certain options.

Microscopy ChannelEnzymeFluorophores Option 1Fluorophores Option 2
S1HRPOpal™ 690nmTSA Vivid 650nm
C2HRPOpal™  520nmTSA Vivid 520nm
C3HRPOpal™  570nmTSA Vivid 570nm
C4HRPOpal™  780nmOpal™ 780nm


Fluorophores required to perform RNAscope smRNA-RNA HD Fluorescent Assay

Fluorophores Part number (ACD)Recommended dilution range
TSA Vivid Fluorophore 520323271 TSA Vivid Fluorophore 520 (ACD)1:750–1:3000
TSA Vivid Fluorophore 570323272 TSA Vivid Fluorophore 5701:750–1:3000
TSA Vivid Fluorophore 650323273 TSA Vivid Fluorophore 6501:750–1:3000


Fluorophores Part number (Akoya Bioscience)Recommended dilution range
Opal 520FP1487001KT: Opal 520 Reagent Pack1:750–1:3000
Opal 570FP1488001KT: Opal 570 Reagent Pack1:750–1:3000
Opal 690FP1497001KT: Opal 690 Reagent Pack1:750–1:3000
Opal Polaris 780FP1501001KT: Opal Polaris 780 Reagent Pack TSA-DIG#  : 1:750–1:3000
Polaris 780: 1:187.5–1:750 


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Advanced Cell Diagnostics

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