Abstract
This study presents a comprehensive protocol for siRNA-mediated knockdown in the differentiation of P19 cells into neuronal-like cells. Utilizing a retinoic acid (RA)-induced neurogenesis model, P19 cells were cultured under specific conditions that facilitated the formation of embryoid bodies (EBs), which were subsequently differentiated into neuronal-like cells. In this investigation, we specifically targeted the Nfe2l1 gene using siRNA transfection to assess the efficiency and effectiveness of our protocol throughout the neuronal differentiation process. Validation of the differentiation was performed through quantitative reverse transcription PCR (RT-qPCR) analysis, measuring the expression levels of key neuronal markers, including Map2 and Pax6 along with the pluripotency marker Oct4. Additionally, the efficiency of the siRNA-mediated knockdown was confirmed by western blot analysis, which demonstrated significant gene silencing at protein levels. These findings underscore the potential of siRNA technology in elucidating gene function during neuronal differentiation and highlight the critical role of targeted gene silencing in advancing neurogenesis research. Furthermore, this study provides a robust and reliable protocol for gene knockdown in neuronal-like cells derived from P19 cells, thereby facilitating further investigations into the intricate molecular mechanisms that govern neurogenesis, neuronal maturation, and overall brain development.•Developed a novel protocol for targeted gene knockdown in P19 cells during neuronal differentiation.•Successful silencing of the Nfe2l1 gene during neuronal differentiation, validated by western blot.•This study provides a reliable protocol for gene knockdown in neuronal differentiation, aiding functional studies of genes in neurogenesis.