RNAscope™ Plus smRNA-RNA LS 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 models. Combining proprietary RNAscope and miRNAscope ISH technologies, we are now introducing the RNAscope Plus smRNA-RNA LS assay which is designed as an automated assay on the Leica Biosystems' BOND RX Research Advanced Staining System.  It 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 detect 1 small RNA along with up to 3 mRNA targets on the Leica Biosystems' BOND RX automated staining system
  • Visualize antisense oligonucleotides (ASOs), microRNAs (miRNAs), small interfering RNAs (siRNAs), and other smaller RNAs that are 17-50 nt in length along with up to 3 mRNA targets
  • Visualize gene regulation with morphological context
  • Validate miRNA biomarkers in intact tissues
  • Assess small RNA therapeutic delivery and biodistribution
  • Add a visual dimension to interrogate heterogeneous tissues and evaluate therapeutic efficacy.
  • Combined ACD patented signal amplification system and the Tyramide Signal Amplification (TSA™) technology




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 LS 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 key technological advantage of this assay is the significant signal boost with simultaneous background noise suppression which is due to advances in ACD’s patented signal amplification technology combined with Tyramide Signal Amplification (TSA™) technology.


Guidelines for Microscope Channel Selection:

RNAscope Plus smRNA-RNA LS 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 Akoya Biosciences.

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


What to order
Visualization requires a multispectral fluorescent imaging system (e.g. Nuance FX Multiplex Biomarker Imaging System)


Order the following components from ACD:

ComponentCat. No.
RNAscope™ Plus smRNA-RNA LS Reagents Kit (Includes RNAscope™ Plus  smRNA-RNA LS Detection Kit and RNAscope LS Multiplex TSA Buffer Pack)322786
miRNAscope LS Target Probes (Catalog or Made-to-Order LS-S1 Probes)Various
RNAscope LS 2.5 Target Probes
(Catalog or Made-to-Order C2, C3 & C4 LS 2.5 Probes)
RNAscope Plus smRNA-RNA 4-plex LS Positive Control Probe-Hs323368
RNAscope Plus smRNA-RNA 4-plex LS Positive Control Probe-Mm323378
RNAscope Plus smRNA-RNA 4-plex LS Positive Control Probe-Rn323388
RNAscope Plus smRNA-RNA 4-plex LS Negative Control Probe323398


Fluorophores required to perform RNAscope smRNA-RNA LS 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 


Required materials and equipment from Leica BOND RX
The RNAscope smRNA-RNA LS Fluorescent Assay requires specific materials and equipment available only from Leica Biosystems.

ComponentCat. No.
Leica Biosystems’ BOND RX System — automated slide stainer
BOND 30 mL Open containers OP309700
BOND 7 mL Open containers* OP79193
BOND Research Detection SystemDS9455
BOND Universal CovertileS21.4611
BOND Epitope Retrieval Solution 1-1L (RTU)AR9961
BOND Epitope Retrieval Solution 2-1L (RTU)AR9640
BOND Dewax Solution – 1L (RTU)AR9222
BOND Wash Solution 10X Concentrate – 1LAR9590
BOND Aspirating Probe Cleaning SystemCS9100
BOND Mixing StationsS21.1971


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