Co-transfection of Plasmid DNA and mRNA

Co-delivery of plasmid DNA and mRNA is a co-transfection technique employed in specific applications such as creating gene insertion using Zinc Finger Nucleases (ZFNs). Following is a straight-forward protocol using TransIT®-mRNA Transfection Kit adapted from Sigma's user manual for CompoZr® Targeted Integration Kit-AAVS (Sigma Aldrich). While this co-transfection protocol has been reported to work with TransIT®-mRNA Transfection Kit, other Mirus' DNA transfection reagents deliver DNA more efficiently. For optimal co-delivery of plasmid DNA and mRNA, we recommend making separate DNA transfection complexes using one of Mirus' broad spectrum DNA transfection reagents such as TransIT-X2® Dynamic Delivery System followed by separate mRNA transfection complexes using TransIT®-mRNA Transfection Kit. This order of addition is due to the time-sensitive nature of mRNA transfection complex formation using TransIT®-mRNA Transfection Kit. Detailed protocol links for each approach are listed below.

Transient DNA & mRNA co-transfection protocol in a 6-well plate using TransIT®-mRNA Transfection Kit
Transient DNA & mRNA co-transfection protocol in a 6-Well Plate using TransIT-X2® System (for DNA delivery) and
TransIT®-mRNA Transfection Kit (for mRNA delivery)

Transient DNA & mRNA Co-Transfection Protocol in a 6-Well Plate using TransIT®-mRNA Transfection Kit

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 using TransIT®-mRNA Kit

  1. Warm TransIT®-mRNA and mRNA Boost reagents 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 a 1 μg/μl stock) RNA. Pipet gently to mix completely.
  4. Add 2.5-10 µg DNA; this might be application specific. For gene integration using CompoZr® Targeted Integration Kit -AAVS1, 8.75 µg (5 μl of a 1.75 μg/μl stock) is recommmended as per manufacturer's protocol.
  5. Add 2.5-7.5 μl mRNA Boost Reagent to the diluted RNA and DNA mixture. Pipet gently to mix completely.
  6. Add 2.5-7.5 μl TransIT®-mRNA Reagent to the diluted RNA and DNA mixture. Pipet gently to mix completely.
  7. Incubate at room temperature for 2-5 minutes to allow sufficient time for complexes to form. Do not incubate the complexes for more than 5 minutes.

NOTE: Depending on the application, the optimal reagent levels to use for RNA and DNA co-transfection should be tested by including a range of TransIT®-mRNA Reagent levels (2.5,5, and 7.5 μl) with a range of mRNA Boost Reagent amounts (2.5, 5, and 7.5 μl). The amounts of RNA and DNA themselves might also need to be optimized to ensure successful co-delivery.

C. Distribute the complex mixtures 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.

NOTE: While the above protocol has been reported to work with TransIT®-mRNA Transfection Kit, we recommend making separate DNA transfection complexes using one of Mirus' broad spectrum DNA transfection reagents such as TransIT-X2® Dynamic Delivery System followed by separate mRNA transfection complexes using TransIT®-mRNA Transfection Kit. This allows optimal co-delivery of plasmid DNA and mRNA; a detailed protocol is listed below.

Transient DNA & mRNA Co-Transfection Protocol in a 6-Well Plate using
TransIT-X2® Dynamic Delivery System and TransIT®-mRNA Transfection Kit

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 DNA transfection complexes using TransIT-X2® Dynamic Delivery System
(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 7.5 µl of TransIT-X2®. Pipet gently to mix completely. For further optimization of your cell type, test additional amounts of TransIT-X2®.
  5. Incubate at room temperature for 15-30 minutes to allow sufficient time for complexes to form.

C. Prepare mRNA transfection complexes using TransIT®-mRNA Kit
(while the DNA transfection complexes are incubating)

  1. Warm TransIT®-mRNA and mRNA Boost reagents 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 a 1 μg/μl stock) RNA. Pipet gently to mix completely.
  4. Add 5 μl mRNA Boost Reagent to the diluted RNA mixture. Pipet gently to mix completely.
  5. Add 5 μl TransIT®-mRNA Reagent to the diluted RNA mixture. Pipet gently to mix completely.
  6. Incubate at room temperature for 2-5 minutes to allow sufficient time for complexes to form. Do not incubate the complexes for more than 5 minutes.

D. Distribute the complex mixtures to cells in complete growth medium

  1. Add the co-transfection complexes (prepared in Step B and C) 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.
 

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