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
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.invitrogen.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.
Protocol - Reverse Transfection
Use this procedure to reverse transfect Stealth™ RNAi or siRNA into mammalian cells in a 24-well format (for other formats, see Scaling Up or Down Transfections). In reverse transfections, the complexes are prepared inside the wells, after which cells and medium are added. Reverse transfections are faster to perform than forward transfections, and are the method of choice for high-throughput transfection.
Optimize transfections as described in Optimizing Transfections, especially if transfecting a mammalian cell line for the first time. All amounts and volumes are given on a per well basis.
- For each well to be transfected, prepare RNAi duplex-Lipofectamine® RNAiMAX complexes as follows.
- Dilute 6 pmol RNAi duplex in 100 µl Opti-MEM® I Medium without serum in the well of the tissue culture plate. Mix gently.
- Mix Lipofectamine® RNAiMAX gently before use, then add 1 µl Lipofectamine® RNAiMAX to each well containing the diluted RNAi molecules. Mix gently and incubate for 10-20 minutes at room temperature.
To qualitatively assess transfection efficiency, we recommend using a K1F11 Stealth™ Select RNAi (available through www.invitrogen.com/rnaiexpress; for human cells, oligo HSS105842 is a good choice). Adherent cells in which K1F11/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.
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.
Rel. surf. area1
Vol. of plating medium
Dilution medium reverse transfection
Dilution medium forward transfection
2 x 10 µl
2 x 20 µl
2 x 50 µl
2 x 250 µl
2 x 500 µl
2 x 1 ml
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 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