• Mar
    21
    Application of combined RNA in situ hybridization and multiplex immunohistochemistry to unravel tumor heterogeneity in prostate cancer
    Nallasivam Palanisamy, PhD, Associate Scientist, Henry Ford Health System
    Courtney Anderson, PhD, Senior Scientist, R&D, Advanced Cell Diagnostics Registration
    Application of combined RNA in situ hybridization and multiplex immunohistochemistry to unravel tumor heterogeneity in prostate cancer
    Nallasivam Palanisamy, PhD
    Associate Scientist, Henry Ford Health System
    Courtney Anderson, PhD
    Senior Scientist, R&D, Advanced Cell Diagnostics Registration
    Outline:

    Prostate cancer is a complex disease with multiple tumors originating independently at different stages of growth. Despite the identification of key morphological differences between individual tumor foci, the underlying molecular mechanisms driving growth within individual foci are often poorly understood. This is because traditional molecular and genetic studies involving systemic sampling of large tumor foci or high Gleason grade tumor foci often miss small tumor foci with important driver aberrations and high metastatic potential. To avoid overlooking foci with important driver aberrations, well-characterized cancer-specific markers can be used to screen the entire prostate tissue to assess molecular differences in individual tumor foci. A clearer understanding of foci-specific molecular heterogeneity is important, as these molecular differences may prognosticate tumor growth and treatment success. 

    In this presentation, we will demonstrate the power of dual immunohistochemistry (IHC) and in situ hybridization (ISH) as a technique to probe multiple cancer-specific molecular markers in the morphological context. Combining the protein detection capabilities of IHC with single-molecule mRNA expression analysis using RNAScope® ISH technology builds a more complete and robust picture of the molecular mechanisms governing individual tumor foci. In addition to highlighting the development of dual IHC/ISH to characterize solid tumor heterogeneity at the molecular level, we will present how high-sensitivity, single RNA molecule detection ISH combined with IHC can be applied to other biological questions to provide valuable information on the expression of molecular markers in nearly any biological pathway.

    Learning Objectives:
    • Applying dual IHC/ISH methods
    • Understanding tumor heterogeneity
    Dates and Registration:
    March
    21
    10:00 AM PT
    1:00 PM ET
    5 PM GMT
    6 PM CET
    Register Now
  • Mar
    23
    Advances in neuroscience research utilizing modern in situ RNA gene expression techniques: Impressions from an early adopter of RNAscope
    Dr. Jerold Chun, MD. Ph.D, Professor & Senior Vice President of Neuroscience Drug Discovery, Sanford Burnham Prebys Medical Discovery Institute
    Advances in neuroscience research utilizing modern in situ RNA gene expression techniques: Impressions from an early adopter of RNAscope
    Dr. Jerold Chun, MD. Ph.D
    Professor & Senior Vice President of Neuroscience Drug Discovery, Sanford Burnham Prebys Medical Discovery Institute
    Outline:

    In situ hybridization (ISH) has become a mainstay of many approaches to understanding gene expression in biology and pathology, whereby labelled probes complementary to mRNA sequences – i.e., anti-sense probes – are used to localize mRNAs in cells and tissue sections. In existence for some 5 decades, the basic ISH technique still requires substantial optimization covering a range of skills, from basic molecular biology to tissue isolation and preparation, and is subject to further variability amongst operators. RNAscope offers a superior alternative and scalable approach to classical ISH, with recent iterations allowing resolution of much shorter targets and even junctional sequences. This webcast provides an introduction to this comparatively new commercial technology and its use in answering questions previously addressed by classical techniques. It is presented from the viewpoint of an ISH user with 30-years of classical experience who has switched to RNAscope and BaseScope, with a particular emphasis on its use in studying the nervous system. As a tangible example, the use of RNAscope was instrumental in validating “hits” identified from interrogating single-neuron transcriptomes obtained by sequencing single neuronal nuclei of the human brain (Science 352, 1586-1590 (2016)), and examples from this and other research efforts will be presented.

    Learning Objectives:

    In this webcast you will learn; Advances in situ hybridization and RNAscope enable RNA gene expression analysis off-the-shelf and single day assays BaseScope gene expression analysis enables splice variance and short target analysis mRNA analysis in neuroscience research where no antibodies are available Detection of long non-coding RNA (lncRNA) RNA-seq to target validation and validation of (cell type-specific) genetic modifications Ability to visualize in situ expression of virtually any gene, any species and any tissue You will also have the opportunity to ask questions of our speaker, live during the broadcast!

    Dates and Registration:
    March
    23
    10 AM PT
    1 PM ET
    5 PM GMT
    6 PM CET
    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.

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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