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

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.

Comparison of partially and fully chemically-modified siRNA in conjugate-mediated delivery in vivo

“…Mouse primary cortical neurons were prepared as previously described ( 22 ). In brief, pregnant C57BL6 females were anesthetized with Avertin (Sigma, #T48402), followed by cervical dislocation.…”

“…Protocols for oligonucleotide treatment have been described in detail in Alterman et al. bioprotocols( 22 ).…”

Nuclear Localization of Huntingtin mRNA Is Specific to Cells of Neuronal Origin

“…Primary cortical neurons were prepared and maintained as described in (Alterman et al, 2017). The procedure was performed using sterile standard dissection tools.…”

“…1 × 10 5 neurons per well were plated on 96-well plates pre-coated with poly-L-lysine (BD BIOCOAT #356515) as described in (Alterman et al, 2017). Cells were processed 7 days post-treatment.…”

“…Htt mRNA silencing was performed as described in (Alterman et al, 2017). mRNA levels were assessed using the QuantiGene 2.0 Assay (Affymetrix #QS0011) and Htt mRNA level was normalized to Ppib mRNA (housekeeping control).…”