Stealth/siRNA Transfection Protocol Lipofectamine 2000

Invitrogen™ Lipofectamine™ 2000 is a proprietary formulation for the transfection of nucleic
acids (DNA and RNA) into eukaryotic cells providing the following advantages:

• Highest transfection efficiency in many cell types and formats ( e .g. 96-well). Refer to the Cell Lines database for a list of cell types successfully transfected.
• Nucleic acid-Lipofectamine 2000 complexes can be added directly to cells in culture medium, in the presence or absence of serum.
• It is not necessary to remove complexes or change/add medium after transfection, but complexes may be removed after 4-6 hours.
  

Guidelines for Transfection

• Use the procedure on page 2 to transfect cells with short interfering RNA (siRNA) or Invitrogen™ Stealth RNAi™.
• Use the procedure on page 3 to transfect cells with plasmid DNA. We recommend Opti-MEM I Reduced Serum Medium (Cat. No. 31985-062) to dilute Lipofectamine 2000 and nucleic acids before complexing.
• Do not add antibiotics to media during transfection as this causes cell death. Maintain the same seeding conditions between experiments.
• Test serum-free media for compatibility with Lipofectamine 2000 since some serum-free formulations ( e .g. CD293, SFM II, VP-SFM) may inhibit cationic lipid-mediated transfection.
  

Note:  If you are performing RNAi experiment, refer to Seven Steps to RNAi Success for more tips. Also, see our section of RNAi protocols.

Use this brief procedure to transfect Stealth RNAi or siRNA into mammalian cells in a 24-well format. All amounts and volumes are given on a per well basis. Use this procedure as a starting point; optimize transfections as described in Optimizing Stealth RNAi or siRNA Transfection, especially if you are transfecting a mammalian cell line for the first time.

1. One day before transfection, plate cells in 500 µl of growth medium without antibiotics such that they will be 30-50% confluent at the time of transfection. Note: Transfecting cells at a lower density allows a longer interval between transfection and assay time, and minimizes the loss of cell viability due to cell
   overgrowth.
   


2. For each transfection sample, prepare oligomer-Lipofectamine 2000 complexes as follows:
   • A. Dilute 20 pmol Stealth RNAi or siRNA oligomer in 50 µl Gibco™ Opti-MEM™ I Reduced Serum Medium without serum (resulting concentration of RNA in Opti-MEM is 400 nM). Mix gently
     
   • B. Mix Lipofectamine 2000 gently before use, then dilute 1 µl in 50 µl Opti-MEM I Reduced Serum Medium. Mix gently and incubate for 5 minutes at room temperature. Note: Proceed to the next step within 25 minutes.
   • C. After the 5-minute incubation, combine the diluted oligomer with the diluted Lipofectamine 2000. Mix gently and incubate for 20 minutes at room temperature (solution may appear cloudy).
           
3. Add the oligomer-Lipofectamine 2000 complexes to each well containing cells and medium. Mix gently by rocking the plate back and forth.

Incubate the cells at 37°C in a CO2 incubator for 24-96 hours until you are ready to assay for gene knockdown. Medium may be changed after 4-6 hours.

Optimizing Stealth RNAi or siRNA Transfection
To obtain the highest transfection efficiency and low non-specific effects, optimize transfection conditions by varying RNA and Lipofectamine 2000 concentrations. Test 10-50 pmol RNA and 0.5-1.5 µl Lipofectamine 2000 for 24-well format. Depending on the nature of the target gene, transfecting cells at higher densities may also be considered when optimizing conditions.

To transfect cells in different tissue culture formats, vary the amounts of Lipofectamine 2000, nucleic acid, cells, and medium used in proportion to the relative surface area, as shown in the table. With automated, high-throughput systems, a complexing volume of 50 µl is recommended for transfections in 96-well plates.

Note: You may perform rapid 96-well plate transfections by plating cells directly into the transfection mix. Prepare complexes in the plate and directly add cells at twice the cell density as in the basic protocol in a 100 µl volume. Cells will adhere as usual in the presence of complexes.

¹  Surface areas may vary depending on the manufacturer.
²  Volumes of dilution medium in Step 2a & 2b of DNA or RNAi transfection protocols.