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    A High-throughput Assay for mRNA Silencing in Primary Cortical Neurons in vitro with Oligonucleotide Therapeutics

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    Authors
    Alterman, Julia F.
    Coles, Andrew H.
    Hall, Lauren M.
    Aronin, Neil
    Khvorova, Anastasia
    Didiot, Marie C.
    UMass Chan Affiliations
    Department of Medicine
    Department of Molecular Medicine
    RNA Therapeutics Institute
    Document Type
    Journal Article
    Publication Date
    2017-08-20
    Keywords
    Branched DNA
    Primary cortical neurons
    QuantiG ene 2.0
    Screening
    siRNA
    Molecular Biology
    Nervous System Diseases
    Neuroscience and Neurobiology
    Research Methods in Life Sciences
    
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    Link to Full Text
    https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5621760/
    Abstract
    Primary neurons represent an ideal cellular system for the identification of therapeutic oligonucleotides for the treatment of neurodegenerative diseases. However, due to the sensitive nature of primary cells, the transfection of small interfering RNAs (siRNA) using classical methods is laborious and often shows low efficiency. Recent progress in oligonucleotide chemistry has enabled the development of stabilized and hydrophobically modified small interfering RNAs (hsiRNAs). This new class of oligonucleotide therapeutics shows extremely efficient self-delivery properties and supports potent and durable effects in vitro and in vivo. We have developed a high-throughput in vitro assay to identify and test hsiRNAs in primary neuronal cultures. To simply, rapidly, and accurately quantify the mRNA silencing of hundreds of hsiRNAs, we use the QuantiGene 2.0 quantitative gene expression assay. This high-throughput, 96-well plate-based assay can quantify mRNA levels directly from sample lysate. Here, we describe a method to prepare short-term cultures of mouse primary cortical neurons in a 96-well plate format for high-throughput testing of oligonucleotide therapeutics. This method supports the testing of hsiRNA libraries and the identification of potential therapeutics within just two weeks. We detail methodologies of our high throughput assay workflow from primary neuron preparation to data analysis. This method can help identify oligonucleotide therapeutics for treatment of various neurological diseases.
    Source

    Bio Protoc. 2017 Aug 20;7(16). doi: 10.21769/BioProtoc.2501. Link to article on publisher's site

    DOI
    10.21769/BioProtoc.2501
    Permanent Link to this Item
    http://hdl.handle.net/20.500.14038/40415
    PubMed ID
    28966945
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    Rights
    Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
    ae974a485f413a2113503eed53cd6c53
    10.21769/BioProtoc.2501
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