Protocol for odorant-binding protein 2A gene knockdown by dual siRNA transfection in a three-dimensional human epidermal equivalent model.

Gene knockdown by small interfering RNA (siRNA) transfection is widely used to investigate gene function. Here, we present a protocol for the knockdown of the odorant-binding protein 2A (OBP2A) gene in a three-dimensional human epidermal equivalent model (3DE-model). We describe steps for growing human epidermal keratinocytes (normal human epithelial keratinocytes [NHEKs]) for 3DE-model construction in monolayer culture. We then detail procedures for transfecting the cells with siRNA, followed by a second siRNA transfection during 3DE-model construction. The efficiency of gene knockdown is verified by qPCR and ELISA. For complete details on the use and execution of this protocol, please refer to Nakanishi et al.¹.