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  • Jul
    14
    2020 July 14 - Viral Detection by ACD’s in situ Hybridization Assay Services
    Mladen Senicar, MS, Research Associate, Advanced Cell Diagnostics
    Jyoti Phatak, Associate Scientist, Applications, Advanced Cell Diagnostics
    2020 July 14 - Viral Detection by ACD’s in situ Hybridization Assay Services
    Mladen Senicar, MS
    Research Associate, Advanced Cell Diagnostics
    Jyoti Phatak
    Associate Scientist, Applications, Advanced Cell Diagnostics
    Outline:

    Nucleic acid-based molecular detection methods have revolutionized viral detection, offering several essential advantages such as sensitivity, specificity and speed. Direct detection of viral RNA in human or other animal cells by RNA in situ hybridization (ISH) is a powerful tool to establish the etiology and pathogenesis of viral disease.

     

    We will discuss detection of AAV and LV vector biodistribution and transgene expression in preclinical animal models with RNAscope® and BaseScope™ in situ hybridization. RNAscope in situ assays of AAV DNA and transgene mRNA combine the molecular sensitivity of qPCR with single-cell resolution and the context of tissue morphology. BaseScope enables specific detection of even single nucleotide differences and can easily differentiate AAV human transgene from non-human primate and other animal ortholog sequences.

     

    In this webinar, we will demonstrate how ACD’s assay service offerings can be leveraged to detect viral infection and pathogenesis, and enhance our understanding of globally important viruses, such as SARS-CoV-2.

    Learning Objectives:
    • Detection of positive single-stranded RNA viruses (Zika, MERS-CoV, hepatitis A/C/E, coronavirus) using RNAscope technology
    • Visualization of the SARS-Cov-2 viral RNA using the V-nCoV2019-S probe and detect viral replication with the V-nCoV2019-S sense
    • Simultaneous AAV vector biodistribution and transgene mRNA expression analysis
    • Localize specific cell types and tissues infected with HIV/SIV – ideal for identifying and measuring persistent reservoirs with single-cell resolution in full morphological context
    Dates and Registration:
    July
    14
    10am PST
    1pm EST
    7pm CEST
    Register Now

You can watch a video or download presentation of the past webinars.

Recorded Webinars

Follow a step-by-step visual guide to assist with running the RNAscope Manual Assay.

How to use EZ-Batch Tray

De-paraffinization

De-paraffinization is performed to ensure complete removal of the paraffin from FFPE samples to allow for the probes to penetrate the target RNA after adequate pretreatment.

Endogenous Peroxidase Blocking

RNAscope H2O2 step is performed during the RNAscope assay to block endogenous peroxidase enzyme activity to prevent hazy background after detection.Note: Pretreatment 1 refers to RNAscope H2O2 reagent and is the first pretreatment perfomed on your samples for Chromogenic assays.

Target Retrieval (Boiling)

Target Retrieval step is a heat induced epitope retrieval method that is necessary to reverse the cross-linking caused by the formalin fixation step. Note: Pretreatment 2 refers to Target Retrieval reagent.For an alternative steamer protocol refer to the appendix of User Manual Part 2 Brown and Red.

Protease Plus (Protease Digestion)

Protease Plus is a broad spectrum protease that is intended to permeabilize the samples adequately to allow the probes to reach the target mRNA.Note : Pretreament 3 refers to Protease Plus reagent.

Target Probe Hybridization

ACD provides properietary double "ZZ" oligo probes designed to hybridize to your specific RNA target 

Amplification (Amp1-Amp6) with Wash Step

RNAscope detection reagents amplify hybridization signals via sequential hybridization of amplifiers.

DAB Colormetric Reaction

Chromogenic detection is based on the enzyme substrate reaction which leads to the formation of an insoluble precipitate visualized in the form of punctate dots for the RNAscope assay.

Gill's Hematoxylin Counter Stain

Hematoxylin staining is a counterstain used to provide a contrast to better visualize the signal and to observe  the morphology of the sample and identify the localization of the signal.

Tissue Dehydration

The tissue dehydration step after the counterstaining step results in removal of excess moisture which provides better  tissue morphology and preservation of the signal.

Mounting

The final step in RNAscope after staining requires the use of  mounting media to adhere a coverslip to tissue section or cell smear. This helps protect the sample and the staining from physical damage and helps improve the clarity and contrast of an image during microscopy.

Download any of the following webinar presentation documents.

PresentationsDownload File
Ready Set Go Getting Started with RNAscope_May 12 2015

Ready Set Go Getting Started with RNAscope_Apr 14 2015

RNAscope Troubleshooting Tips_June 16 2015

RNAscope Troubleshooting Tips_July 14 2015

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Please contact us directly, by phone or email, for any technical assistance with the RNAscope® assay or relevant products.

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