__NOTOC__[[Category: Cell Culture ]][[Category: Knockdown]] ==siRNA Double Transfection of RAW 264.7 Materials==*Cells with Lipofectamine 2000 in a 6-Well Dish==AfCS Procedure Protocol PP00000262Version 1, 10/12/04well dishThe 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 postInvitrogen cat# 11668-transfection or from protein isolated 72 hr post-transfection019 for 1.5mL) ===Day 1=Required Amounts==#Count RAW 264.7 cells and plate in a 6-*Calculate RNA to transfect (typically 40 pmol per well plate at 5 x 105 cells/well, 2.5 ml/well.)#Incubate cells at 37 °C *Lipofectamine(in a humidified, 5% CO2 incubator for 24 hr.===Day 2==uL) =RNA(in pmoles) * 50#In an RNase-free tube, dilute 10 μl of 40 μM siRNA duplex *OptiMEM(in a total volume of 250 μl Opti-MEM. Mix gently.#Mix Lipofectamine 2000 gently before use.#In a separate RNase-free tubeuL, dilute 10 μl equal for DNA and Lipofectamine 2000 ) = Lipofectamine(in a total volume of 250 μl Opti-MEM. Mix gently and incubate for 5 min at room temperature.uL) * 50#After the 5 min incubation, combine the diluted duplex with the diluted Lipofectamine 2000 ==Protocol (total volume is 500 μl6 well dish). Mix gently and incubate for 20 min at room temperature.==#During Plate cells the 20 min incubation, aspirate medium day before from cells. Add 500 μl fresh RAW 264.7 growth medium 1 (RAWGM1) a confluent dish diluted to cells.#Add Lipofectamine 2000 complexes to wells (500 μl/well). The final siRNA concentration is 400 nM in a final volume of 1 ml:12 into DMEM/well. Mix gently by rocking the plate, and incubate cells for 4 hour at 37 °C in a humidified, 5% CO2 incubator.FBS with no PSG#After 4 hr, add 1.5 ml RAWGM1 to For each well.8. Continue to incubate cells at 37 °C in a humidifiedprepare, 5% CO2 incubator.===Day 3===scalin up as required:#In an RNase-free tube, dilute 10 μl of #100uL OptiMEM plus 40 μM duplex in a total volume pmoles of 250 μl Opti-MEM. Mix gentlyRNA.#Mix #100uL OptiMEM plus 2 uL of Lipofectamine 2000 gently before use.#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 #Incubate ~5 min minutes at room temperature.#After #Combine the 5 min incubation, combine the diluted duplex with the diluted two volumes of OptiMEM/DNA and OptiMEM/Lipofectamine 2000 (total volume is 500 μlshould be 200uL/well). Mix gently and incubate for ##Incubate ~20 min at room temperature.#During the 20 min incubation, aspirate medium from cells. Add 500 μl fresh RAWGM1 to cells.#Add DNA/Lipofectamine 2000 complexes to wells (500 μl/well~200 uL). The final siRNA concentration is 400 nM in a final volume to the 1 mL of 1 ml/well. Mix gently by rocking media on the plate, and incubate cells for 4 hr at 37 °C in a humidified, 5% CO2 incubator.#After 4 hr, add 1.5 ml RAWGM1 Gently rock plate to each well.mix#Continue to incubate After ~4h re-feed cells at 37 °C in a humidified, 5% CO2 incubatorwith normal media.===Day 4===#Harvest cells for RNA analysis as indicated in Cell Lysis and Total RNA Isolation Protocol adapted from RAW 264.7 Cells, AfCS protocol PP00000260, at approximately 48 hr from start of first transfection.#For cells destined for protein analysis, change medium on cells.#Aspirate medium and add 2.5 ml fresh medium per well.===Day 5===#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.==Reagents and Materials==*Cell culture cluster, 6 well[http: Corning Inc//tools.; catalog noinvitrogen. 3516*Microtube, 1com/content/sfs/manuals/lipofectamine2000_man.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[[ Category:Cell Culture ]][[ Category:Transfection]pdf Product Manual]
