A Highly Efficient siRNA Transfection Method in Primary Cultured Cortical Neurons.

Abstract

Transfecting neurons remains technically challenging due to their sensitivity. Conventional methods, such as Lipofectamine 2000 or Lipofectamine RNAiMAX, often result in significant cytotoxicity, which limits their utility. Although lentiviral transfection offers high efficiency, it is hindered by high costs and complex procedures. This experiment employs a small interfering RNA (siRNA)-specific transfection reagent from the Kermey company. This reagent is a novel nanoparticle-based lipid material designed for the efficient delivery of oligonucleotides, including siRNA, into a wide range of cell types. Its efficacy in achieving high transfection efficiency in neurons, however, has not yet been established. After several days of in vitro neuronal culture, researchers can perform a simple transfection procedure using this reagent to achieve robust transfection efficiency. Notably, the protocol does not require medium replacement 6-8 h post-transfection, streamlining the workflow and minimizing cellular stress. Key features • Based on Kermey's siRNA-specific transfection reagent, we present a method for efficient in vitro transfection of siRNA into primary cultured mouse cortical neurons. • No observable adverse effects are detected in the transfected neurons during the entire experiment. • This method enables consistent and efficient knockdown of the target protein. • Phosphoglycerate dehydrogenase (PHGDH) siRNA and siNC (negative control) siRNA can be transfected into neuronal cells after 72 h of in vitro culture.