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Tips from the Bench: Transfection Tip

Co-transfection Protocol for Plasmid DNA and siRNA/miRNA

Co-transfection of plasmid DNA and siRNA/miRNA is a technique popularly used by cell biologists, particularly to assess 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.

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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, 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. 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 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® 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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