RNAscope Plus

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

 

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
S1 HRP Opal™ 690nm TSA Vivid 650nm
C2 HRP Opal™  520nm TSA Vivid 520nm
C3 HRP Opal™  570nm TSA Vivid 570nm
C4 HRP Opal™  780nm Opal™ 780nm

 

Fluorophores required to perform RNAscope smRNA-RNA HD Fluorescent Assay

Fluorophores Part number (ACD)Recommended dilution range
TSA Vivid Fluorophore 520 323271 TSA Vivid Fluorophore 520 (ACD) 1:750–1:3000
TSA Vivid Fluorophore 570 323272 TSA Vivid Fluorophore 570 1:750–1:3000
TSA Vivid Fluorophore 650 323273 TSA Vivid Fluorophore 650 1:750–1:3000

 

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

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