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Reverse Transfection of siRNA using Lipofectamine RNAiMAX in a 24-well plate format

Use this procedure to reverse transfect Stealth RNAi or siRNA into mammalian cells in a 24-well format. 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.

1. 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.
2. Dilute cells in complete growth medium without antibiotics so that 500 µl contains the appropriate number of cells to give 30-50% confluence 24 hours after plating. Use 20,000-50,000 cells/well for suspension cells.
3. To each well with RNAi duplex - Lipofectamine™ RNAiMAX complexes, add 500 µl of the diluted 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.
4. Incubate the cells 24-72 hours at 37°C in a CO2 incubator until you are ready to assay for gene knockdown.

Reverse Transfection of siRNA using Lipofectamine 2000 in a 96 well plate format

• This is plucked straight from Invitrogen's own protocol. Scale up for transfection in larger culture vessels.

Introduction

Lipofectamine 2000 Reagent is a proprietary formulation that facilitates highly efficient delivery of Stealth RNA molecules or short interfering RNA (siRNA) to mammalian cells for RNAi analysis. Here, we describe reverse transfection using Lipofectamine 2000. Reverse transfection is a method for high throughput (HTP) transfection of multiple siRNA or Stealth RNAi duplexes, and is well suited in combination with RNAi duplexes pre-dispensed in 96-wells plates, such as the Stealth RNAi Human Collections (available through our website, www.invitrogen.com).

Reverse transfection combines RNA, transfection reagent, and cells in an altered sequence compared to traditional Lipofectamine 2000 transfection protocols. In short, a different RNAi molecule is put in each well prior to transfection and combined with diluted Lipofectamine 2000 to form complexes in each well. Cells are added directly to the Lipofectamine 2000-RNA complexes and transfection occurs while cells are attaching to the well.

General Guidelines for Transfection

Follow these general guidelines when using Lipofectamine 2000 to reverse transfect siRNA or Stealth RNAi duplexes into mammalian cells.

• Use low-passage cells, and make sure that cells are healthy and greater than 90% viable before transfection. This protocol is for use with adherent cells, as transfection efficiency for suspension cells generally is lower and may need optimization.
• Do not add antibiotics to the medium during transfection as this reduces transfection efficiency and causes cell death.
• For optimal results, use Opti-MEM I Reduced Serum Medium to dilute Lipofectamine 2000, DNA, and dsRNA oligomers prior to complex formation.
• To increase accuracy and reduce assay variability, we recommend performing triplicate transfections for each sample condition.

Materials Needed

You should have the following materials on hand before beginning:

• Mammalian cell line cultured in the appropriate growth medium
• Stealth RNAi or siRNA of interest (20 μM stock in 1X RNA Annealing/Dilution Buffer)
• RNAi controls
• Lipofectamine 2000 Reagent (Catalog nos. 11668-027 or 11668-019; store at +4°C and mix gently before use)
• Opti-MEM I Reduced Serum Medium (pre-warmed; Catalog nos. 31985-062 or 31985-070)
• 96-well tissue culture plates and tissue culture supplies

Transfection Procedure

Use this procedure to reverse transfect your siRNA or Stealth RNAi duplexes into mammalian cells in 96-wells plates using Lipofectamine 2000. The complexes are prepared inside the wells, after which cells and medium are added. Remember to include the proper positive and negative controls in your experiment.

1. For each well to be transfected, prepare DNA-RNAi molecule-Lipofectamine 2000 complexes as follows.
   • Mix LipofectamineTM 2000 gently before use, then dilute 0.25 μl Lipofectamine 2000 in 25 μl Opti-MEM I Medium without serum in a separate vessel. Mix gently and incubate for 5 minutes at room temperature.
   • Dilute 15 pmol siRNA or StealthTM RNAi in 25 μl Opti-MEM I Medium without serum in the well of the tissue culture plate. Mix gently.
   • After the 5 minute incubation, add the diluted Lipofectamine 2000 to the wells with the diluted RNAi molecules. Mix gently and incubate for 15 minutes at room temperature to allow complex formation to occur. The solution may appear cloudy, but this will not impede the transfection.
2. Add 100 μl complete growth medium without antibiotics with 20,000 cells to each well containing RNAi molecule-Lipofectamine 2000 complexes. This gives a final volume of 150 μl and a final RNA concentration of 100 nM. Mix gently by rocking the plate back and forth.
3. Incubate the cells at 37°C in a CO2 incubator until you are ready to harvest cells and assay for your target gene.

Harvest cells 24-48 hours after transfection to assay for target gene knockdown.