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Rnascope system

Manufactured by Advanced Cell Diagnostics
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The RNAscope system is a highly sensitive and specific in situ hybridization technology for detecting and quantifying RNA expression in individual cells within intact tissue sections. The system enables the visualization and localization of target RNA molecules with high specificity and signal-to-noise ratio.

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Lab products found in correlation

Cm3050, by Leica (3 mentions) 4 mm biopsy punch, by Integra LifeSciences (3 mentions) Proprietary probes, by Advanced Cell Diagnostics (2 mentions) Axio zoom v16 microscope, by Zeiss (2 mentions) Sodium pentobarbital, by Vortech Pharmaceuticals (1 mentions) Cm1850 cryostat, by Leica (1 mentions) Superfrost slide, by Thermo Fisher Scientific (1 mentions) Colorfrost plus slides, by Thermo Fisher Scientific (1 mentions) Rnascope 2.5 universal pretreatment reagents, by Advanced Cell Diagnostics (1 mentions) Sc 7819, by Santa Cruz Biotechnology (1 mentions) A11055, by Thermo Fisher Scientific (1 mentions) Model fv1000, by Olympus (1 mentions) Alexa fluor 488 donkey anti goat, by Thermo Fisher Scientific (1 mentions) Cobas 4800 system, by Roche (1 mentions) Dm6000, by Leica (1 mentions) Aquatex, by Merck Group (1 mentions) Superfrost plus slides, by Avantor (1 mentions) Rabbit anti cgrp, by Merck Group (1 mentions) Prolong gold antifade, by Thermo Fisher Scientific (1 mentions) Opal 620 reagent, by Akoya Biosciences (1 mentions)

28 protocols using rnascope system

1

High-Risk HPV Detection in Adenosquamous Carcinomas

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HPV detection for high-risk HPV subtypes was performed on adenosquamous carcinomas in the tissue microarray that had sufficient tissue to score and had not been improperly fixed or stored (n=23). HPV in situ hybridization with a chromogen was performed using the Advanced Cell Diagnostics (Hayward, CA) RNAscope® system (catalogue no.312598). The RNAscope® Probe “HPV HR18” contains probes targeting E6 and E7 mRNA for the following high-risk subtypes: HPV16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 73, and 82. The methodology and interpretation were discussed in detail in a previous paper (18 (link)). Positive and negative control cases were used for optimal results. A full range of cytoplasmic and nuclear signals were encountered. Cases were interpreted to be HPV-positive if any brown signal appeared (nuclear, cytoplasmic) in the presence of a negative control.
Stolnicu S., Hoang L., Hanko-Bauer O., Barsan I., Terinte C., Pesci A., Aviel-Ronen S., Kiyokawa T., Alvarado-Cabrero I., Oliva E., Park K.J, & Soslow R.A. (2018). Cervical Adenosquamous Carcinoma: Detailed Analysis of Morphology, Immunohistochemical Profile, and Outcome in 59 cases. Modern pathology : an official journal of the United States and Canadian Academy of Pathology, Inc, 32(2), 269-279.
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2

Oxytocin Receptor Expression Detection

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In situ hybridization was performed using the RNAscope system (Advanced Cell Diagnostics) following the manufacturer’s protocol. Pre-treatment consisted of dehydration, followed by incubation with hydrogen peroxide and protease IV at room temperature. The Multiplex Fluorescent Kit v2 protocol was followed using commercial probes for the OT receptor (Oxtr, NM_001081147.1, #402658-C3). Images were captured by Zeiss 880 inverted confocal microscopy. Visualized cells with more than 5 puncta per cell were classified as positive neurons.
Ba X., Ran C., Guo W., Guo J., Zeng Q., Liu T., Sun W., Xiao L., Xiong D., Huang Y., Jiang C, & Hao Y. (2022). Three-Day Continuous Oxytocin Infusion Attenuates Thermal and Mechanical Nociception by Rescuing Neuronal Chloride Homeostasis via Upregulation KCC2 Expression and Function. Frontiers in Pharmacology, 13, 845018.
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3

Spinal Cord RNAscope Profiling in Mice

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Male or female mice were euthanized by IP injection of sodium pentobarbital (30 mg/kg; Vortech Pharmaceuticals) then transcardially perfused with 0.01M phosphate buffer (PB) followed by 4% paraformaldehyde (PFA) dissolved in PB. Lumbar spinal cords were dissected out and post-fixed for an additional 2 hours in room-temperature 4% PFA. Tissue was transferred to RNAse-free 30% sucrose in 0.1M phosphate buffered saline (PBS) overnight. 14 μm transverse sections were cut on a CM 1850 cryostat (Leica) and mounted onto Superfrost slides (Fisher). Slides were dried for 20 min at −20°C, then stored at −80°C.
The RNAscope system (Advanced Cell Diagnostics) was used per manufacturer’s directions for fresh-frozen tissue pretreatment, except for omission of the initial on-slide fixation step. Probe hybridization and detection was then carried out using Multiplex Fluorescent Kit v2, following manufacturer’s directions. The following RNAscope probes were used: Adra2b (425321), Cartpt (432001), Crhr1 (418011-C2), Gucy2d (425451-C2), Npbwr1 (547181), Pdyn (318771, 318771-C2), and Pnoc (437881). TSA Plus Cyanine 3 and Cyanine 5 system (PerkinElmer #NEL744E001KT, #NEL745E001KT) were used to visualize fluorescent in situ signals. TSA fluorophores were diluted in RNAscope TSA buffer (Cy3 1:1500, Cy5 1:1000).
Serafin E.K., Chamessian A., Li J., Zhang X., McGann A., Brewer C.L., Berta T, & Baccei M. (2019). Transcriptional profile of spinal dynorphin-lineage interneurons in the developing mouse. Pain, 160(10), 2380-2397.
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4

Spatial Expression of Immune Genes

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Mice were deeply anesthetized with isoflurane and transcardially perfused with 25 ml PBS followed by 25 ml 4% paraformaldehyde/1% picric acid. Following perfusion, L3-L5zen spinal cords or DRGs were isolated and post-fixed overnight at 4°C in the same fixative. Tissues were subsequently washed several times in PBS, followed by cryopreservation using a sucrose gradient. tissues were then embedded in OCT medium (Tissue-Tek) and cryosectioned to produce 14 μm-thick sections, which were mounted onto charged slides. Each tissue block (and thus, each slide) contained both WT and KO tissues to account for any variability in staining between slides, and to control for the specificity of RNAscope probes targeting STING (Tmem173) or Ifnar1. In situ hybridization was performed using the RNAscope system (Advanced Cell Diagnostics) in accordance with the manufacturer’s instructions, using a protocol tailored to the Multiplex Fluorescent Kit v2. We used probes directed against murine Ifnar1 (catalog 512971, NM_010508.2) and murine STING (Tmem173; catalog 413321, NM_028261.1). Following the completion of the RNAscope protocol, immunohistochemistry was performed as described in the next section. The in situ hybridization experiments were repeated in three independent experiments.
Donnelly C.R., Jiang C., Andriessen A.S., Wang K., Wang Z., Ding H., Zhao J., Luo X., Lee M.S., Lei Y.L., Maixner W., Ko M.C, & Ji R.R. (2021). STING controls nociception via type-I interferon signaling in sensory neurons. Nature, 591(7849), 275-280.
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5

High-Risk HPV Subtype Detection in GAS Cases

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HPV detection for high-risk HPV subtypes was performed on all GAS cases (n = 62). HPV in situ hybridization with a chromogen was performed using the RNAscope system from Advanced Cell Diagnostics (Catalog no. 312598) following the manufacturer's instruction. The RNAscope Probe "HPV HR18" contains probes targeting E6 and E7 mRNA for the following 18 high-risk subtypes: HPV16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 73, and 82 .
Yang J., Peng Y., Ding Y., Liu Y., Wang Y., Liu Y, & Liu C. (2024). The Clinicopathological and Molecular Characteristics of Endocervical Gastric-Type Adenocarcinoma and the Use of Claudin18.2 as a Potential Therapeutic Target. Modern pathology : an official journal of the United States and Canadian Academy of Pathology, Inc, 37(10).
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6

Quantitative HPV RNA Expression Analysis

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The RNAscope scoring system was used to examine each specimen, as described in previous studies [23 (link), 24 (link)]. HPV fluorescent in situ hybridization was performed using a chromogen and the RNAscope system (Advanced Cell Diagnostics; catalog no. 312598). The RNAscope probe HPV HR18 contains probes that target E6 and E7 mRNA in these high-risk subtypes: HPV16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 73, and 82. The RNAscope results were classified into five degrees based on the following scoring guidelines: 0, no staining or <  1 dot in 10 cells (40x); 1, 1–3 dots in each cell (20–40x); 2, 4–10 dots in each cell as well as a few dot clusters (20–40x); 3, > 10 dots in each cell as well as dot clusters in < 10% positive cells (20x); 4, > 10 dots in each cell as well as dot clusters in > 10% positive cells (20x). An RNAscope score ≥ 1 indicated positivity.
Zhang S.W., Luo R.Z., Sun X.Y., Yang X., Yang H.X., Xiong S.P, & Liu L.L. (2021). Co-expression of SOX2 and HR-HPV RISH predicts poor prognosis in small cell neuroendocrine carcinoma of the uterine cervix. BMC Cancer, 21, 332.
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7

Multiplexed in situ Hybridization of Neuronal Transcripts

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Multiplexed in situ hybridization to indicated transcripts were performed using the RNAscope system (Advanced Cell Diagnostics). Whole brains from injected rodents were flash-frozen in OCT medium (Tissue Tek) using a dry ice/ethanol bath at 10-15 days post-injection. Cortical tissue from marmoset visual cortex was collected using a 4 mm biopsy punch (Integra) and immediately flash-frozen in OCT. All samples were cryosectioned at 12 μm (Leica CM3050S) and processed according to probe manufacturer instructions. Briefly, fixed and dehydrated sections were co-hybridized with proprietary probes (Advanced Cell Diagnostics) to neuronal marker transcripts, followed by differential fluorescence tagging. Signals in cells identified using DAPI staining were co-localized on an AXIOZoom V16 microscope (Zeiss).
Mehta P., Kreeger L., Wylie D.C., Pattadkal J.J., Lusignan T., Davis M.J., Turi G.F., Li W.K., Whitmire M.P., Chen Y., Kajs B.L., Seidemann E., Priebe N.J., Losonczy A, & Zemelman B.V. (2019). Functional Access to Neuron Subclasses in Rodent and Primate Forebrain. Cell reports, 26(10), 2818-2832.e8.
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8

Multiplexed in situ Hybridization of Neuronal Transcripts

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Multiplexed in situ hybridization to indicated transcripts were performed using the RNAscope system (Advanced Cell Diagnostics). Whole brains from injected rodents were flash-frozen in OCT medium (Tissue Tek) using a dry ice/ethanol bath at 10-15 days post-injection. Cortical tissue from marmoset visual cortex was collected using a 4 mm biopsy punch (Integra) and immediately flash-frozen in OCT. All samples were cryosectioned at 12 μm (Leica CM3050S) and processed according to probe manufacturer instructions. Briefly, fixed and dehydrated sections were co-hybridized with proprietary probes (Advanced Cell Diagnostics) to neuronal marker transcripts, followed by differential fluorescence tagging. Signals in cells identified using DAPI staining were co-localized on an AXIOZoom V16 microscope (Zeiss).
Mehta P., Kreeger L., Wylie D.C., Pattadkal J.J., Lusignan T., Davis M.J., Turi G.F., Li W.K., Whitmire M.P., Chen Y., Kajs B.L., Seidemann E., Priebe N.J., Losonczy A, & Zemelman B.V. (2019). Functional Access to Neuron Subclasses in Rodent and Primate Forebrain. Cell reports, 26(10), 2818-2832.e8.
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9

HPV Subtype Detection in Tissue Microarrays

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HPV detection for HR-HPV subtypes was performed on all ECAs in the TMA that had sufficient tissue to score, and had not been improperly fixed or stored (n=168). HPV in-situ hybridization with a chromogen was performed using the Advanced Cell Diagnostics (ACD) (Hayward, CA) RNAscope® system (catalogue no.312598). The RNAscope® Probe “HPV HR18” contains probes targeting E6 and E7 mRNA for the following high-risk subtypes: HPV16,18,26,31,33,35,39,45,51,52,53,56,58,59,66,68,73 and 82. The methodology and interpretation were discussed in detail in a previous paper.17 (link)
Stolnicu S., Barsan I., Hoang L., Patel P., Chiriboga L., Terinte C., Pesci A., Aviel-Ronen S., Kiyokawa T., Alvarado-Cabrero I., Pike M.C., Oliva E., Park K.J, & Soslow R.A. (2018). Diagnostic algorithmic proposal based on comprehensive immunohistochemical evaluation of 297 invasive endocervical adenocarcinomas. The American journal of surgical pathology, 42(8), 989-1000.
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10

Histopathological Analysis of Perch Tissues

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During a complete necropsy of the 11 fish, we used 3 whole perch for histologic examination. We fixed these fish in 10% buffered formalin for 24 hours and embedded cut sections of the gills, longitudinal head sections, and longitudinal cut sections of the body cavity in paraffin. We prepared 3-μm sections and stained them with hematoxylin and eosin according to standard protocols. We conducted chromogenic in situ hybridization on all formalin-fixed paraffin-embedded (FFPE) tissues used for histopathology. We performed staining with the RNAscope system (Advanced Cell Diagnostics, https://acdbio.com) (Appendix).
Hierweger M.M., Koch M.C., Rupp M., Maes P., Di Paola N., Bruggmann R., Kuhn J.H., Schmidt-Posthaus H, & Seuberlich T. (2021). Novel Filoviruses, Hantavirus, and Rhabdovirus in Freshwater Fish, Switzerland, 2017. Emerging Infectious Diseases, 27(12), 3082-3091.
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