Co-transfection of Plasmid DNA and siRNA/miRNA

Co-transfection of plasmid DNA and siRNA/miRNA is a technique popularly used by cell biologists, for applications such as knockdown rescue or siRNA/miRNA mediated knockdown of gene expression from a plasmid DNA that is being delivered to the cell. Generally, transfection reagents that can deliver smaller nucleic acids such as siRNA/miRNA to the cytoplasm efficiently are not as successful in plasmid DNA delivery to the nucleus. This is due to the size/charge difference between siRNA/miRNA and plasmid DNA, and the subcellular location for those molecules to act (cytoplasm for siRNA/miRNA and nucleus for plasmid DNA). The new TransIT-X2® Dynamic Delivery System affords cutting-edge co-delivery of plasmid DNA and siRNA/miRNA due to its novel, non-liposomal, polymeric nature.

For a detailed protocol for co-transfecting plasmid DNA and siRNA/miRNA, please click on the link below:

Transient DNA & siRNA Co-Transfection Protocol in a 6-Well Plate using TransIT-X2® System

Functional Co-transfection of Plasmid DNA and siRNA Using TransIT-X2 System

Functional Co-transfection of Plasmid DNA and siRNA Using the TransIT-X2® Dynamic Delivery System. TransIT-X2® Dynamic Delivery System was used to transfect plasmid Cy®5 labeled DNA encoding nuclear YFP and Cy®3 labeled siRNA into HeLa cells. Transfection was performed in 6-well plates with Poly-L-Lysine (PLL) coated coverslips using 4 µl of TransIT-X2® to deliver 2 µg of DNA and 25 nM siRNA (2:1 reagent:DNA ratio). Actin cytoskeleton was stained using Alexa Fluor® 350 Phalloidin. Images (63X) were captured at 24 hours post-transfection using a Nikon A1R confocal microscope. Image key: yellow (nuclear YFP), blue (Cy®5 labeled DNA), red (Cy®3 labeled siRNA), green (actin cytoskeleton).

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

  1. 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.
  2. Incubate the cells overnight.

B. Prepare co-transfection complexes (Immediately before transfection)

  1. Warm TransIT-X2® to room temperature and vortex gently before using.
  2. Place 250 µl of Opti-MEM® I Reduced-Serum Medium in a sterile tube.
  3. Add 2.5 µg (2.5 µl of 1 µg/µl stock) plasmid DNA. Pipet gently to mix completely.
  4. Add 6.8 µl of a 10 µM siRNA stock solution (25 nM final concentration per well). Pipet gently to mix completely.
  5. 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®.
  6. 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

  1. Add the co-transfection complexes (prepared in Step B) drop-wise to different areas of the wells.
  2. Gently rock the culture vessel back-and-forth and from side-to-side to evenly distribute the co-transfection complexes.
  3. Incubate for 24-72 hours or as required. It is not necessary to replace the complete growth medium with fresh medium.
  4. Harvest cells and assay for knockdown of target gene expression.
 

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