RNAiMAX Forward Transfections Lipofectamine

Lipofectamine® RNAiMAX is a proprietary formulation specifically developed for the transfection of siRNA and Stealth™ RNAi duplexes into eukaryotic cells. Lipofectamine® RNAiMAX provides the following advantages:

• High transfection efficiencies in many cell types to minimize background expression from untransfected cells and maximize knockdown
• Minimal cytotoxicity to reduce non-specific effects and cellular stress
• Generally requires low concentrations of RNAi duplexes to obtain high knockdown levels, further minimizing non-specific effects
• A broad peak of optimal transfection activity with minimal cytotoxicity, allowing achievement of high knockdown levels despite differences in cell density, minor pipetting inaccuracies, and other variations

Important Guidelines for Transfection

• Reverse transfection and forward transfection protocols can be used for most cell lines tested. Cell-type specific transfection protocols are available at www.lifetechnologies.com/RNAi or through Technical Service.
• We recommend Opti-MEM® I Reduced Serum Medium (Cat. No. 31985-062) to dilute RNAi duplexes and Lipofectamine® RNAiMAX before complexing.
• Do not add antibiotics to media during transfection as this causes cell death. Test serum-free media for compatibility with Lipofectamine® RNAiMAX.
• To assess transfection efficiency, we recommend using a KIF11 Stealth™ Select RNAi, as described in Assessing Transfection Efficiency.
• Use 10 nM RNAi duplex and indicated procedure as a starting point; optimize transfections as described in Optimizing Transfections.

Use this procedure to forward transfect Stealth™ RNAi or siRNA into mammalian cells in a 24 well format (for other formats, see Scaling Up or Down Transfections, page 4) . In forward transfections, cells are plated in the wells, and the transfection mix is generally prepared and added the next day. Optimize transfections as described in Optimizing Transfections, (page 3), especially if transfecting a mammalian cell line for the first time. All amounts and volumes are given on a per well basis.
Note: For some cell lines (e.g. MCF-7 or HepG2), we recommend reverse transfections.

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 
2. For each well to be transfected, prepare RNAi duplex-Lipofectamine® RNAiMAX complexes as follows:
    

• Dilute 6 pmol RNAi duplex in 50 µl Opti-MEM® I Reduced Serum Medium without serum. Mix gently.
• Mix Lipofectamine™ RNAiMAX gently before use, then dilute 1 µl in 50 µl Opti-MEM® I Reduced Serum Medium. Mix gently.
• Combine the diluted RNAi duplex with the diluted Lipofectamine™ RNAiMAX. Mix gently and incubate for 10-20 minutes at room temperature.


3. Add the RNAi duplex-Lipofectamine® RNAiMAX complexes to each well containing cells. This gives a final volume of 600 µl and a final RNA concentration of 10 nM. Mix gently by rocking the plate back and forth.>/li>
4. Incubate the cells 24-48 hours at 37°C in a CO₂ incubator until you are ready to assay for gene knockdown. Medium may be changed after 4-6 hours.

To qualitatively assess transfection efficiency, we recommend using a KIF11 Stealth™ Select RNAi (available through www.lifetechnologies.com/rnaiexpress; for human cells, oligo HSS105842 is a good choice).  Adherent cells in which KIF11/Eg5 is knocked down exhibit a "rounded-up" phenotype after 24 hours due to mitotic arrest (Weil, D. et al. Biotechniques 2002, 33: 1244-1248); slow growing cells may take up to 72 hours.  Alternatively, growth inhibition can be assayed after 48-72 hours. Note: The BLOCK-iT™ Fluorescent Oligo (Cat. No 2013) is optimized for use with Lipofectamine® 2000, and is not recommended for Lipofectamine® RNAiMAX.

Optimizing Transfections

To obtain the highest transfection efficiency and low non-specific effects, optimize transfection conditions by varying RNAi duplex and Lipofectamine® RNAiMAX concentrations. Test 0.6-30 pmol RNAi duplex (final concentration 1-50 nM) and 0.5-1.5 µl Lipofectamine® RNAiMAX for 24-well format. For extended time course experiments (> 72 hours), consider a cell density that is 10-20% confluent 24 hours after plating.
Note:  The concentration of RNAi duplex required will vary depending on the efficacy of the duplex.

To transfect cells in different tissue culture formats, vary the amounts of Lipofectamine® RNAiMAX, RNAi duplex, cells, and medium used in proportion to the relative surface area, as shown in the table.

¹ Surface areas may vary depending on the manufacturer.
² If the volume of Lipofectamine® RNAiMAX is too small to dispense accurately, and you cannot pool dilutions, predilute Lipofectamine® RNAiMAX 10-fold in Opti-MEM® I Reduced Serum Medium, and dispense a 10-fold higher amount (should be at least 1.0 µl per well). Discard any unused diluted Lipofectamine® RNAiMAX.

For cotransfections of plasmid DNA and Stealth™ RNAi or siRNA into mammalian cells, we recommend using Lipofectamine® 2000 (Catalog no. 11668-027), which is superior for plasmid transfections. If you want to use Lipofectamine® RNAiMAX for your cotransfections, perform a reverse transfection as described on page 2 with the following modifications:

1: Add 20 ng (for 24-well format) of plasmid DNA to the diluted RNAi duplex.
 
2: Add cells such that they will be 80-100% confluent 24 hours after plating.