The process of intentionally introducing purified or naked nucleic acid into eukaryoit cells is known as transfection. This is usually done through nonviral methods (Al-Rubeai 2014). In addition, it is also used in research to modulate and study gene expression. Consequently, transfection techniques act as an diagnostic instrument that facilitates the categorization of protein synthesis, cell growth and development and generic functions. Transfection assays do not only give room for the encroachment of cellular study, but may also enhance strategies for drug discovery (Love, 2016).
Some of the materials required include Eppendorf tubes, Opti-MEM® Reduced Serum Medium and Plasmid DNA (0.5 – 5µg / µL stock). When choosing a transfection reagent, it is recommended that one identify the culture conditions and the cell type of the assay. According to Zhang & Surapaneni (2012) primary cells, neurons and rare cell cultures are normally difficult to transect hence the need to use reagents to help in the transfection facilitation even when transfecting such cells may be challenging. Some common types of reagents include; Lipofectamine 3000, Lipofectamine RNAiMAX, Lipofectamine MessengerMAX, Lipofectamine 2000, Neon Electroporation, Invivofectamine 3.0. Conn (2012) also list some of the materials required during the transfection process, such as;, MicroPulserTM electroporator, TransFectin lipid reagent, SiLentFect lipid reagent, Gene Pulser MXcell TM electroporation system, HeliosTM gene gun, PDS-1000/HeTM biolistic particle delivery system and Gene Pulser Xcell TM electroporation system (Conn, 2012).
Lipofectamine procedure does involve seven recommended steps. The first is to seed cells to be 70-90% confluent at transfection, the main component are the adherent cells. The second step involves diluting four amounts of Lipofectamine®Reagent in Opti-MEM® Medium, the components are Opti-MEM® Medium and Lipofectamine2 2000 Reagent (Basché, Müllen & Schmidt 2014). The third step requires dilution of DNA in Opti- MEM® Medium, the components needed are Opti-MEM® Medium and DNA (0.5-5µg/µL).
The fourth step requires the addition of diluted DNA to diluted Lipofectamine® 2000 Reagent in a ratio of one to one. The recommended components are diluted DNA Total and diluted Lipofectamine® 2000 reagent. The fifth stage involves incubation, which normally takes five minutes at room temperature. According to Kim (2017) the sixth step requires the addition of DNA- lipid complex to cells. This stage requires, DNA-lipid complex per well, final DNA used per well and final Lipofectamine® 2000 reagent used per well. Lastly, the final step involves visualization and analysis of transfected cells. During this stage incubation of cells should last between one to three days at 37 degrees Celsius thereafter the transfected cells can be analyzed (Kim, 2017).
Step one takes place on day zero. Step two to step six takes place in day one while step seven starts from step 2-4 (Stamm, Smith & Lührmann 2012).
- Al-Rubeai, M. (2014) Animal cell culture.
- Basché, T., MüLlen, K., & Schmidt, M. (2014). From single molecules to nanoscopically structured materials.
- Conn, P. M. (2013). Methods in enzymology. trafficking and oligomerization Volume five hundred and twenty one, Volume five hundred and twenty one.
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- Kim, B. W. (2017). Clinical regenerative medicine in urology. Singapore, Springer.
- Love, J. C. (2016). Micro-and nanosystems for biotechnology. Wiley-Blackwell.
- Stamm, S., Smith, C. W. J., & LüHrmann, R. (2012). Alternative pre-mRNA splicing: theory and protocols. Weinheim, Wiley-Blackwell.
- Zhang, D., & Surapaneni, S. (2012). ADME-Enabling Technologies in Drug Design and Development. Hoboken, John Wiley & Sons.