Applications: Co-transfection

Co-transfection of Multiple Plasmid DNA, siRNA or mRNA

Co-transfection of multiple nucleic acids is a technique increasingly employed by researchers. Applications that popularly utilize multiple plasmid DNA co-transfection are virus production, protein-protein interaction studies, stable cell line generation, or simple addition of reporter DNA constructs to normalize experimental output. Multiple siRNA molecules can be transfected for pooled siRNA transfection. Recently, co-delivery of multiple mRNA transcripts encoding differentiation factors has been employed for non-integrative stem cell reprogramming applications. One example utilizing co-transfection of multiple plasmid DNA molecules for virus production is illustrated below.

For a detailed protocol for co-transfecting multiple plasmid DNAs, please click on the following link:

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

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High Titer Virus Production Using TransIT-2020

Rescue of Influenza Virus by Reverse Transfection With TransIT®-2020 Transfection Reagent. 293T and MDBK cells are reverse transfected with up to 10 separate influenza virus rescue plasmids per experiment. Reagent:total DNA complexes are prepared with TransIT®-2020 Transfection Reagent at a 3:1 ratio of reagent to total DNA. Virus-containing supernatants are harvested 48-96 hr post-transfection. Titers from transfected cells can be as high as 107 pfu/ml. Rescued virus is then amplified on MDBK cells to increase titer and total yield. Viral stocks are subsequently titered by plaque assay on MDCK cells. Titers from representative rescue experiments with different viral strains and reassortants are shown. This experimental strategy is adapted from Neumann, et al. 2005 PNAS.

Data courtesy of Andrew Mehle, University of Wisconsin-Madison

All of the TransIT® broad-spectrum and cell-type specific DNA transfection reagents can be used for co-transfecting multiple plasmid DNAs. For siRNA co-transfection, any of the TransIT® broad-spectrum siRNA transfection reagents can be used. Depending on the cell type being transfected, one reagent may have superior performance over others; for cell-type specific recommendations, please consult the Reagent Agent® transfection database. Multiple mRNAs can be transfected using the broad spectrum TransIT®-mRNA Transfection Kit.

Nucleic Acid(s) to be Co-transfected Transfection Reagent Application(s)
Multiple plasmid DNAs Option 1: TransIT-X2® Dynamic Delivery System
Option 2: TransIT®-2020 Transfection Reagent
Option 3: TransIT®-LT1 Transfection Reagent
Virus production
Dual reporter assays
Stable cell line generation
Multiple siRNA/miRNAs Option 1: TransIT-X2® Dynamic Delivery System
Option 2: TransIT-TKO® Transfection Reagent
Option 3: TransIT-siQUEST® Transfection Reagent
Pooled siRNA transfection
Multiple mRNAs TransIT®-mRNA Transfection Kit Stem cell reprogramming
Multiple oligos TransIT®-Oligo Transfection Reagent Pooled oligo transfection

In addition to general user protocol guidelines such as optimal cell confluence, high quality nucleic acid, etc., following guidelines should be considered for co-transfection experiments of similar nucleic acid molecules.

  • Ratio of Transfection Reagent: Total Nucleic Acid: It is critical to maintain the same amount of total nucleic acid as the user protocol recommends and to divide the total nucleic acid into a mixture of the different nucleic acids that need to be transfected. Depending on the application, the ratio of each nucleic acid may need further optimization to achieve the best experimental results. For example, during stable cell line generation, co-transfection of a plasmid mixture containing 5 to 10 parts gene expression plasmid and 1 part antibiotic selection marker plasmid is generally sufficient to ensure that the selected cells will express both the gene of interest and the selection marker.
  • Premixing of Nucleic Acids: It is also important to prevent preferential complexation of one nucleic acid over the other(s). This can be accomplished by completely premixing the different nucleic acids before adding the transfection reagent. The order of addition of different nucleic acids should not affect on the transfection outcome if the nucleic acid mixture is mixed completely before forming the transfection complex.

Outlined below is an easy-to-follow co-transfection protocol for multiple plasmid DNAs using TransIT-X2® Dynamic Delivery System in a 6-well format. TransIT-X2® Dynamic delivery system can also be used to co-deliver multiple siRNAs; additionally, it also enables efficient co-delivery of plasmid DNA and siRNA (detailed protocol here). For additional details, please refer to the user protocol of TransIT-X2® System.

For co-delivery tips for other nucleic acid molecules such as mRNA, oligos, etc., please contact

Transient DNA 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 total plasmid DNA. Plasmid DNAs can be added sequentially depending on the optimized ratio. For example, if generating stable cell lines using an antibiotic selection marker on a separate plasmid than the gene of interest, add 2.25 µg plasmid DNA containing the "gene of interest" first to Opti-MEM® I Reduced-Serum Medium; pipet gently to mix completely. Then, add 0.25 µg of the plasmid DNA containing the "antibiotic selection marker" to this mixture; pipet gently to mix completely.
  4. 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® as per the user protocol. For cell type specific protocol recommendations, please refer to the TransIT-X2® protocol optimization card.
  5. 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 target and/or reporter gene expression.

* The above protocol can be used when using other DNA transfection reagents such as TransIT®-2020 and TransIT®-LT1. However, depending on the transfection reagent and the cell type used for co-transfection, the ratio of the transfection reagent: total DNA may need to be re-optimized.

Technical Resources

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