Custom RNA in situ hybridization probes were developed to detect the full-length E6/E7 mRNA sequence of MmuPV1 by Advanced Cell Diagnostics for use with their RNAscope assay kit. CISH was performed according to the manufacturer's instructions for RNAscope2.0 as previously described (37 (link)). Briefly, formalin-fixed, paraffin-embedded (FFPE) mouse tail tissue sections were pretreated with heat and protease prior to hybridization with the probe. If treated with RNase A (Qiagen), slides were incubated for 30 min at room temperature with RNase A in PBS (10 mg/ml). Slides were subsequently washed three times in diethyl pyrocarbonate (DEPC)-treated water for 5 min per wash. To ensure RNA integrity and assay procedures, adjacent sections were also hybridized with a probe for the endogenous housekeeping gene ubiquitin and the bacterial gene dapB (negative control). After washing, a horseradish peroxidase (HRP)-based amplification system was then used to detect the target probes, followed by color development with 3,3′-diaminobenzidine (DAB).
Rnascope assay kit
Manufactured by Advanced Cell Diagnostics
Sourced in United States
The RNAscope assay kit is a platform developed by Advanced Cell Diagnostics for the detection and visualization of RNA molecules in tissue samples. The kit utilizes a proprietary signal amplification technology to enable sensitive and specific detection of target RNA transcripts within the morphological context of cells and tissues.
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5 protocols using rnascope assay kit
1
MmuPV1 E6/E7 mRNA Detection by RNAscope
2
SIV Detection in Macaque Brain
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SIV RNA detection in macaque brain tissue samples (frontal cortex, basal ganglia, and thalamus) was performed using an RNAscope assay kit from Advanced Cell Diagnostics. Custom probes for SIVmac239 were used in conjunction with RNAscope 2.5 HD Detection kit (Brown) according to the manufacturer’s instructions. To detect/quantify the number of SIV+ cells, the entire section was scanned frame by frame for the presence of probe-positive cells.
3
Single-Molecule RNA Detection via RNAScope
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An RNAScope assay was performed to detect single‐molecule RNA using the RNAScope Assay Kit (Advanced Cell Diagnostics, CA). Sixteen paired double‐Z oligonucleotide probes targeting 141–1381 nt of DUXAP9 were designed by ACD. The experiment was performed according to the manufacturer's instructions.
4
Dual-Color FISH Protocol for RNAscope
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FISH was performed using RNAscope assay kit (Advanced Cell Diagnostics, Newark, CA, USA). Mice were transcardially perfused with 4% paraformaldehyde (w/v) in phosphate buffer (0.1 M, pH 7.4), and brains were removed and post-fixed overnight. Then the brains were dehydrated by 10, 20, and 30% sucrose gradient solutions in sequence, until the brain completely sink to the bottom. Brain sections (12 μm) were cut in a cryostat (Leica, Germany). All subsequent steps were performed according to the manufacturer’s instructions of RNAscope assay kit. Probes used: RNAscope® Probe-Mm-Nos1, RNAscope® Probe-EGFP-C2. Finally, these two probes were visualized by Cy3 and Cy5, respectively. RNAscope® 3-plex negative control probe was used to confirm the specificity of the labeling.
5
Single-Molecule RNA Detection by RNAScope Assay
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RNAScope assay was performed to detect the single-molecule RNA using the RNAScope® Assay Kit (Advanced Cell Diagnostics, CA, USA). Fourteen paired double-Z oligonucleotide probes targeting 2–1152 nt of DILA1 were designed using the custom software (Hs-MIR99AHG-O1, NPR-0007680). The experiment was performed according to the manufacturer’s instructions. In brief, cultured cells were fixed by 10% neutral formalin at room temperature for 30 min, incubated in a hydrogen peroxide solution for 15 min, and digested in protease III solution for 20 min. The cells were then hybridized with target probes at 40 °C for 2 h in a hybridization oven. After signal amplification, the cells were conjugated with TSA Plus Cy3 fluorescence at 40 °C for 30 min and blocked by the HRP blocker. Then the cells were counterstained with DAPI and observed by confocal microscopy.
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