In addition to observing general guidelines for transfection such as optimal cell confluence, high quality DNA and siRNA/miRNA, optimal ratio of the reagent:nucleic acid, etc., the following factors should additionally be considered when co-transfecting plasmid DNA and siRNA. The same factors are important when co-transfecting plasmid DNA and other smaller nucleic acids such as miRNAs and miRNA mimics.
- Proper Controls: It is critical to include proper controls to ensure successful delivery of both plasmid DNA and siRNA/miRNA. We recommend transfecting a plasmid only control and a non-specific siRNA only control to verify gene expression from the transfected plasmid DNA and specificity of the siRNA, respectively.
- Premixing of Nucleic Acids: It is very important to prevent preferential complexation of the plasmid DNA over siRNA/miRNA or vice versa. This can be accomplished by completely premixing the plasmid DNA and siRNA before adding the transfection reagent.
Outlined below is an easy-to-follow co-transfection protocol for plasmid DNA and siRNA using TransIT-X2® Dynamic Delivery System in a 6-well format. For other tissue culture formats, refer to the user protocol. This protocol can also be followed when co-transfecting plasmid DNA and miRNA by simply substituting miRNA for siRNA.
Transient DNA & siRNA Co-Transfection Protocol in a 6-Well Plate using
TransIT-X2® Dynamic Delivery System
A. Plate cells
- Approximately 18-24 hours before transfection, plate cells using the following guidelines. For most cell types, cultures should be ≥80% confluent at the time of transfection. For adherent cells, plate cells at a density of 2-6 × 105 cells/well. For suspension cells, plate cells at a density of 8-10 × 105 cells/well.
- Incubate the cells overnight.
B. Prepare co-transfection complexes (Immediately before transfection)
- Warm TransIT-X2® to room temperature and vortex gently before using.
- Place 250 µl of Opti-MEM® I Reduced-Serum Medium in a sterile tube.
- Add 2.5 µg (2.5 µl of 1 µg/µl stock) plasmid DNA. Pipet gently to mix completely.
- Add 6.8 µl of a 10 µM siRNA stock solution (25 nM final concentration per well). Pipet gently to mix completely.
- Add 7.5 µl of TransIT-X2®. Pipet gently to mix completely. For further optimization of your cell type, test additional amounts of TransIT-X2®.
- Incubate at room temperature for 15-30 minutes to allow sufficient time for complexes to form.
C. Distribute the complex mixture to cells in complete growth medium
- Add the co-transfection complexes (prepared in Step B) drop-wise to different areas of the wells.
- Gently rock the culture vessel back-and-forth and from side-to-side to evenly distribute the co-transfection complexes.
- Incubate for 24-72 hours or as required. It is not necessary to replace the complete growth medium with fresh medium.
- Harvest cells and assay for knockdown of target gene expression.