Alterman Julia F, Coles Andrew H, Hall Lauren M, Aronin Neil, Khvorova Anastasia, Didiot Marie-Cécile
RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, MA, USA.
Department of Molecular Medicine, University of Massachusetts Medical School, Worcester, MA, USA.
Bio Protoc. 2017 Aug 20;7(16). doi: 10.21769/BioProtoc.2501.
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 and . We have developed a high-throughput 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.
原代神经元是用于鉴定治疗神经退行性疾病的治疗性寡核苷酸的理想细胞系统。然而，由于原代细胞的敏感性质，使用传统方法转染小干扰RNA（siRNA）既费力又效率低下。寡核苷酸化学的最新进展使得稳定化和疏水修饰的小干扰RNA（hsiRNA）得以开发。这类新型寡核苷酸疗法具有极高的自递送效率，并能产生强大而持久的效果。我们开发了一种高通量检测方法，用于在原代神经元培养物中鉴定和测试hsiRNA。为了简单、快速且准确地定量数百种hsiRNA的mRNA沉默效果，我们使用QuantiGene 2.0定量基因表达检测法。这种基于96孔板的高通量检测方法可以直接从样品裂解物中定量mRNA水平。在此，我们描述一种在96孔板中制备小鼠原代皮质神经元短期培养物的方法，用于寡核苷酸疗法的高通量测试。该方法仅需两周就能支持对hsiRNA文库的检测以及潜在治疗药物的鉴定。我们详细介绍了从原代神经元制备到数据分析的高通量检测工作流程的方法。该方法有助于鉴定用于治疗各种神经疾病的寡核苷酸疗法。
