Protocol for odorant-binding protein 2A gene knockdown by dual siRNA transfection in a three-dimensional human epidermal equivalent model
Summary: 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 g...
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Elsevier
2025-03-01
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| Series: | STAR Protocols |
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| Online Access: | http://www.sciencedirect.com/science/article/pii/S2666166725000322 |
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| author | Shinobu Nakanishi |
| author_facet | Shinobu Nakanishi |
| author_sort | Shinobu Nakanishi |
| collection | DOAJ |
| description | Summary: 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 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. |
| format | Article |
| id | doaj-art-5938b1b8da304f5ebb9b91c9f7d7a6fb |
| institution | Kabale University |
| issn | 2666-1667 |
| language | English |
| publishDate | 2025-03-01 |
| publisher | Elsevier |
| record_format | Article |
| series | STAR Protocols |
| spelling | doaj-art-5938b1b8da304f5ebb9b91c9f7d7a6fb2025-02-07T04:48:21ZengElsevierSTAR Protocols2666-16672025-03-0161103626Protocol for odorant-binding protein 2A gene knockdown by dual siRNA transfection in a three-dimensional human epidermal equivalent modelShinobu Nakanishi0Shiseido Global Innovation Center, Yokohama 220-0011, Japan; Corresponding authorSummary: 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 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.http://www.sciencedirect.com/science/article/pii/S2666166725000322Cell cultureCell-based AssaysGene Expression |
| spellingShingle | Shinobu Nakanishi Protocol for odorant-binding protein 2A gene knockdown by dual siRNA transfection in a three-dimensional human epidermal equivalent model STAR Protocols Cell culture Cell-based Assays Gene Expression |
| title | Protocol for odorant-binding protein 2A gene knockdown by dual siRNA transfection in a three-dimensional human epidermal equivalent model |
| title_full | Protocol for odorant-binding protein 2A gene knockdown by dual siRNA transfection in a three-dimensional human epidermal equivalent model |
| title_fullStr | Protocol for odorant-binding protein 2A gene knockdown by dual siRNA transfection in a three-dimensional human epidermal equivalent model |
| title_full_unstemmed | Protocol for odorant-binding protein 2A gene knockdown by dual siRNA transfection in a three-dimensional human epidermal equivalent model |
| title_short | Protocol for odorant-binding protein 2A gene knockdown by dual siRNA transfection in a three-dimensional human epidermal equivalent model |
| title_sort | protocol for odorant binding protein 2a gene knockdown by dual sirna transfection in a three dimensional human epidermal equivalent model |
| topic | Cell culture Cell-based Assays Gene Expression |
| url | http://www.sciencedirect.com/science/article/pii/S2666166725000322 |
| work_keys_str_mv | AT shinobunakanishi protocolforodorantbindingprotein2ageneknockdownbydualsirnatransfectioninathreedimensionalhumanepidermalequivalentmodel |