Lipofectamine Mediated Knockdown

AfCS Procedure Protocol PP00000262 Version 1, 10/12/04 The AfCS is utilizing RNA interference (RNAi) to manipulate signaling protein expression in the RAW 264.7 macrophage-like cell line. One approach to this technique is transient transfection of gene-specific small interfering RNAs (siRNAs). The following procedure involves the transfection of siRNA into RAW 264.7 cells using Lipofectamine 2000. Cells are transfected with siRNA twice (on subsequent days). Target gene knockdown is assessed from total RNA isolated 48 hr post-transfection or from protein isolated 72 hr post-transfection.

Day 1

1. Count RAW 264.7 cells and plate in a 6-well plate at 5 x 105 cells/well, 2.5 ml/well.
2. Incubate cells at 37 °C in a humidified, 5% CO2 incubator for 24 hr.

Day 2

1. In an RNase-free tube, dilute 10 μl of 40 μM siRNA duplex in a total volume of 250 μl Opti-MEM. Mix gently.
2. Mix Lipofectamine 2000 gently before use.
3. In a separate RNase-free tube, dilute 10 μl Lipofectamine 2000 in a total volume of 250 μl Opti-MEM. Mix gently and incubate for 5 min at room temperature.
4. After the 5 min incubation, combine the diluted duplex with the diluted Lipofectamine 2000 (total volume is 500 μl). Mix gently and incubate for 20 min at room temperature.
5. During the 20 min incubation, aspirate medium from cells. Add 500 μl fresh RAW 264.7 growth medium 1 (RAWGM1) to cells.
6. Add Lipofectamine 2000 complexes to wells (500 μl/well). The final siRNA concentration is 400 nM in a final volume of 1 ml/well. Mix gently by rocking the plate, and incubate cells for 4 hour at 37 °C in a humidified, 5% CO2 incubator.
7. After 4 hr, add 1.5 ml RAWGM1 to each well.

8. Continue to incubate cells at 37 °C in a humidified, 5% CO2 incubator.

Day 3

1. In an RNase-free tube, dilute 10 μl of 40 μM duplex in a total volume of 250 μl Opti-MEM. Mix gently.
2. Mix Lipofectamine 2000 gently before use.
3. In a separate RNase-free tube, dilute 10 μl Lipofectamine 2000 in a total volume of 250 μl Opti-MEM. Mix gently and incubate for 5 min at room temperature.
4. After the 5 min incubation, combine the diluted duplex with the diluted Lipofectamine 2000 (total volume is 500 μl). Mix gently and incubate for 20 min at room temperature.
5. During the 20 min incubation, aspirate medium from cells. Add 500 μl fresh RAWGM1 to cells.
6. Add Lipofectamine 2000 complexes to wells (500 μl/well). The final siRNA concentration is 400 nM in a final volume of 1 ml/well. Mix gently by rocking the plate, and incubate cells for 4 hr at 37 °C in a humidified, 5% CO2 incubator.
7. After 4 hr, add 1.5 ml RAWGM1 to each well.
8. Continue to incubate cells at 37 °C in a humidified, 5% CO2 incubator.

Day 4

1. Harvest cells for RNA analysis as indicated in Cell Lysis and Total RNA Isolation from RAW 264.7 Cells, AfCS protocol PP00000260, at approximately 48 hr from start of first transfection.
2. For cells destined for protein analysis, change medium on cells.
3. Aspirate medium and add 2.5 ml fresh medium per well.

Day 5

1. Harvest cells for protein analysis as indicated in Cell Lysis and Protein Extraction from RAW 264.7 Cells, AfCS protocol PP00000258, at approximately 72 hr from start of first transfection.

• Cell culture cluster, 6 well: Corning Inc.; catalog no. 3516
• Microtube, 1.5 ml, RNase-, DNase-, pyrogen-free: VWR; catalog no. KT749510-1590
• Opti-MEM I reduced-serum medium (1X) liquid: Invitrogen; catalog no. 31985088
• Lipofectamine 2000: Invitrogen; catalog no. 11668019
• RAW 264.7 growth medium 1 (RAWGM1): AfCS Solution Protocol PS00000510