Introducing the RNAscope™ Plus small RNA-RNA Fluorescent Assay

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Combining detection of small regulatory RNA with target RNAs

Visualize Any RNARNAscope™ technology is an advanced in situ hybridization assay that allows for the visualization of single-molecule gene expression directly in intact cells/tissues with single cell resolution. Detection of small RNAs (smRNA) requires a robust, highly specific and sensitive assay with quantifiable signal. The new RNAscope™ Plus smRNA-RNA Fluorescent Assay leverages the RNAscope patented core technology that enables signal amplification and background suppression for multiplex visualization of smRNA expression with target mRNAs in in formalin-fixed paraffin-embedded (FFPE) samples and fresh frozen samples.

The RNAscope Plus smRNA-RNA automated Leica or manual Fluorescent Assays use a proprietary method of in situ hybridization to simultaneously visualize one smRNA target microRNAs (miRNAs), PIWI-interacting RNAs (piRNAs), short interfering RNAs (siRNAs), or antisense oligonucleotides (ASOs)) along with three RNA targets.

Key Features • Unlock the potential to detect small regulatory RNAs

along with mRNA targets.

• Visualize ASOs, siRNAs, miRNAs, piRNAs and other elusive smRNA sequences with the signal boosting tyramide amplification system (TSA) chemistry.

• Interrogate smRNA biomarkers/therapeutics with cell- specific/morphology markers.

• Assess smRNA therapeutic delivery and biodistribution.

• Add a visual dimension to interrogate heterogeneous tissues and evaluate therapeutic efficacy.

Figure 1. RNAscope Plus smRNA-RNA Fluorescent Assay workflow. Starting with properly prepared samples, sections are first pretreated, and then smRNA and RNA-specific probes are hybridized to target RNAs. The RNAscope Plus smRNA-RNA Fluorescent Assay employs four independent signal amplification systems each using a different fluorophore. The assay can provide visualization of transcript expression as well as the positional relationship among four different genes within a cellular context. Single RNA transcripts for each target gene appear as punctate dots that are visible using a fluorescent microscope.

The new RNAscope™ Plus smRNA-RNA Fluorescent Assay workflow

For Research Use Only. Not for use in diagnostic procedures. For Research Use Only. Not for use in diagnostic procedures.

2-week turn around time for custom designs

Universal for virtually any gene and species

Specific and sensitive with performance reliability

Ease of use with ready to use formulations

Over 42,000 unique pre-designed probes available to select from

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RNU6

RNU6

POLAR2A

POLAR2A

PPIB

PPIB

UBC

DAPI/Scramble/dapB

Figure 2. RNAscope™ Plus smRNA-RNA assay performance with positive control probes. Positive control targets, miR-RUN6, PPIB, POLAR2A and UBC/18S were detected across different sample types, A, HeLa cells, B, FFPE mouse colon tissue. DapB probe was used along with a scramble miRNA probe as a negative control. Nuclei were counter-stained with DAPI.

Figure 2B.

For Research Use Only. Not for use in diagnostic procedures.

The new RNAscope™ Plus smRNA-RNA Fluorescent Assay demonstrates robust detection of positive control miRNA and RNA targets in HeLa cells and mouse colon FFPE tissue

Figure 2A.

For Research Use Only. Not for use in diagnostic procedures.

RNU6 miRNA POLAR2A PPIB 18S RNU6

POLAR2A PPIB

18S DAPI/Scramble/dapB

UBC

DAPI

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Expression of miR-205 and associated tumor target genes in head and neck cancer tissue

Figure 4.miR-205

miR-205

TP53

TP53

PanCK

PanCK

PTEN

PTEN

Figure 4. Colocalization of miRNA and associated mRNA targets in Head and Neck cancer. 4-plex smRNA-RNA assay using 1 miRNA (miR-205) and 3 mRNAs targets (PanCK, PTEN and TP53) in tumor. MiRNA-205 was expressed in cancer cells across the tumor. Nuclei were counterstained with DAPI.

miR-124 POLAR2A PPIB

miR-124 POLAR2A PPIB

For Research Use Only. Not for use in diagnostic procedures For Research Use Only. Not for use in diagnostic procedures.

Tissue-specific detection of miR-124 in mouse brain tissue using the RNAscope™ Plus smRNA-RNA Fluorescent Assay

Figure 3A.

Figure 3B.

miR-124

miR-124

UBC

UBC

Figure 3. Specificity of RNAscope™ Plus smRNA-RNA staining. 4-plex smRNA-RNA assay using 1 miRNA (miR-124) specific to brain and 3 mRNAs (POLAR2A, PPIB, UBC). A) Specific staining of miR-124 across cerebellum of mouse brain along with other positive control probes, B) Mouse liver tissue showing absence of miRNA-124 but presence of the positive control targets demonstrating specificity of the assay. Nuclei were counterstained with DAPI.

DAPI

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Figure 5. Tissue specific expression of smRNAs, miR-124/miR-SNORD85 and cell specific mRNAs in mouse brain. 4-plex smRNA-RNA assay using 1 miRNA (miR-124 /miR-SNORD85) and 3 cell type specific mRNAs (Rbfox3, Gfap and Aif3). A) Mouse cerebellum showing specific staining of miR-SNORD85 in the outer layer of the mouse brain cerebellum along with neuronal marker (Rbfox3), astrocytic marker (Gfap) and microglial marker (Aif1). B) Mouse cerebellum showing specific staining of miR-124a along with neuronal marker (Rbfox3), astrocytic marker (Gfap) and microglial marker (Aif1) using the new fluorescent vivid dyes. Nuclei were counterstained with DAPI.

Co-detection of brain specific miRNA and mRNAs using the new fluorescent Vivid dyes™

miR-124

miR-124

miR-SNORD85

miR-SNORD85

Rbfox3

Rbfox3

Rbfox3

Rbfox3

Gfap

Gfap

Gfap

Gfap

Aif1

Aif1

Aif1

Aif1

Figure 5A. Figure 5B.

For Research Use Only. Not for use in diagnostic procedures. For Research Use Only. Not for use in diagnostic procedures.

DAPI DAPI

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Recommended TSA dye combinations for a 4-plex assay

Reconstitute all Opals (except Opal Polaris 780) with 75 µL Dimethylsulfoxide (DMSO). Reconstitute Opal Polaris 780 with 300 µL double distilled water (ddH2O).

Reconstitute all TSA Vivid dyes with 100 µL Dimethylsulfoxide (DMSO). Reconstitute Opal Polaris 780 with 300 μL double distilled water (ddH2O)

Reconstitute all Opals (except Opal Polaris 780) with 75 µL Dimethylsulfoxide (DMSO). Reconstitute Opal Polaris 780 with 300 µL double distilled water (ddH2O).

RNAscope Plus smRNA-RNA Reagent Kits

Component Part number (ACD, Bio-Techne)

Assay Compatibility

RNAscope™ Plus smRNA-RNA HD Reagents Kit 322785 For Manual workflow

RNAscope™ Plus smRNA-RNA LS Reagents Kit 322786 For Leica BOND RX workflow

Fluorophores (Option 2) Part number (Akoya Biosciences) 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 620 FP1495001KT: Opal 620 Reagent Pack* 1:750–1:3000

Opal 690 FP1497001KT: Opal 690 Reagent Pack* 1:750–1:3000

Fluorophores (Option 3) Part number (Akoya Biosciences) 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

Fluorophores (Option 1) Part number (ACD, Bio-Techne and Akoya Biosciences)

Recommended dilution range

TSA Vivid Fluorophore 520 323271 1:750–1:3000

TSA Vivid Fluorophore 570 323272 1:750–1:3000

TSA Vivid Fluorophore 650 323273 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

Made-to-order RNAscope target probe can accommodate your specific needs:

If your gene of interest is not listed in our catalog, ACD can design and manufacture new in situ hybridization probes for you, we term these “RNAscope™ Made-to-order Target Probes”

Any Species Any Tissue

Any Gene

Product Information:

For Research Use Only. Not for use in diagnostic procedures. For Research Use Only. Not for use in diagnostic procedures.

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