Are you new to transfection and don’t know where to get started? Our team of Ph.D. educated Scientific Support Scientists have put together their top suggestions for transfection success. By following this workflow, you can select the right reagents for your needs, then optimize conditions to achieve the best possible results.

Step 1: Choose the right reagents for your experiments

Every cell line is different and may require specific transfection conditions. Therefore, it is important to consider the following factors:

  1. Is the cell type easily transfectable?This is where a brief literature review can come in handy. Start with our Cell Type Guide, or look to journal articles to see if others have successfully transfected the cell type you are studying, and how this was accomplished. If the cells are not transfectable, it may be time to consider electroporation, nucleofection, or another strategy.
  2. What are you transfecting? There are specific transfection reagents for different nucleic acid formats. There are transfection reagents designed for small RNAs, large plasmids, or even co-transfection of a small RNA with a large plasmid, mRNA or protein.

If cells are difficult to transfect with a lipid-based reagent and cannot be electroporated, there are other options. Our Accell siRNAs are chemically modified to self-deliver into hard to transfect cell types without the need for lipid-based transfection reagents or electroporation. Lentiviral delivery of molecules is also a possibility for some applications.

Step 2: Optimize your transfection conditions

Once you have selected the ideal delivery reagent for your cell type, it’s time to determine the optimal delivery conditions. The primary determinants of a successful transfection are transfection reagent volume and cell density. Test a range of lipid volumes using three different cell densities, using an appropriate positive control and quantitative downstream assay, similar to seen in this Tech Note. Our product-specific delivery protocols provide guidance for initial volumes of lipid to test. It is important to monitor cell viability using a resazurin assay (or similar), as lipid-based transfection reagents can be toxic to some cell types. For most applications, an optimized transfection is considered to be >80% delivery efficiency and cell viability.

By following these two steps, you will be able to pick the optimal transfection reagents for your studies and determine the best conditions for your experiments. If you run into trouble or have any questions, please contact our Scientific Support team!

Authors: Ivonne Rubio Ph.D. and Kurt Marshall Ph.D. | Scientific Support

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