However, this method is associated with the risk of pathogenic expression and neoplastic transformation. Among biological methods, viral vectors are used for gene delivery with high transfection efficiency, and can be used for in vivo applications. However, the reagents are expensive and may be toxic to certain target cells. This method requires no special equipment. Lipofection is a chemical method, in which a cationic lipid is employed to mediate the transfer of genes in the target cells. In electroporation, a physical method, genes are transferred into cells using high electric field pulse, and it is commonly used because of the short treatment time and high efficiency however, this technique damages the target cells. Traditional gene transfection methods are of three major types, namely, physical, chemical, and biological. It is a fundamental tool used in medicine and biology, such as in gene therapy, regenerative medicine, drug development, and plant breeding, and therefore, it has to be efficient and practically feasible. Gene transfection is a technique of introducing nucleic acids into cells for characterization of gene function. This may explain how MDP simultaneously achieves high transfection efficiency with minimal cell damage. This implies that plasma characteristics can be adjusted according to target cell requirements, and the transfection process can be optimized with minimum damage to cells and maximum efficiency. In plasma-mediated gene transfection, alterations in electrical and chemical factors can independently regulate plasmid DNA adhesion and triggering of endocytosis, respectively. Gene transfer by plasma irradiation depends mainly on endocytosis, which accounts for at least 80% of the transfer, and clathrin-mediated endocytosis is a dominant endocytosis. This indicates that MDP irradiation supplies sufficient concentrations of ROS, and the stimulation intensity of the electric field determines the transfection efficiency in our system. The electric field threshold required for transfection was approximately 1 kV m −1 in our MDP system. This indicates that plasma-mediated gene transfection utilizes the synergy between electrical and chemical factors. Our results show that both electrical and chemical factors are necessary for gene transfer inside cells by microplasma irradiation. In this study, we used laser-produced plasma to demonstrate that gene transfer does not occur in the absence of electrical factors. Studies revealed that the N-acetylcysteine-mediated inhibition of reactive oxygen species (ROS) activity completely abolished gene transfer. We have developed a new micro-discharge plasma (MDP)-based gene transfection method, which transfers genes into cells with high efficiency and low cytotoxicity however, the mechanism underlying the method is still unknown.