pcDNA™3.1 (+) Mammalian Expression Vector

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	siRNA1 (5′-AGCGCAAGAAGGCGGUGCUTT-3′), siRNA2 (5′-GAGGAUCUGGUGUUUAUCUTT-3′) and siRNA3 (5′-GGUGUCAUCAAGGUGUUCATT-3′) siRNA2 had a more potent silencing effect compared with that of siRNA1 and siRNA3. Add diluted siRNA to diluted Lipofectamine Reagent   Incubate for 5 min at RT and add to cells.  Replace medium after 6h and re-incubate cells for 3 days at 37°C and analyse cells at 24,48 and 72 h.

Protocol tips
siRNA1 (5′-AGCGCAAGAAGGCGGUGCUTT-3′), siRNA2 (5′-GAGGAUCUGGUGUUUAUCUTT-3′) and siRNA3 (5′-GGUGUCAUCAAGGUGUUCATT-3′) siRNA2 had a more potent silencing effect compared with that of siRNA1 and siRNA3. Add diluted siRNA to diluted Lipofectamine Reagent   Incubate for 5 min at RT and add to cells.  Replace medium after 6h and re-incubate cells for 3 days at 37°C and analyse cells at 24,48 and 72 h.

Publication protocol

The sequences of CFL1-small interfering (si)RNA duplexes and the high expression plasmid pcDNA3.1-CFL1 were synthesized by GenePharma Co. Ltd (Shanghai, China). siRNA1 (5′-AGCGCAAGAAGGCGGUGCUTT-3′), siRNA2 (5′-GAGGAUCUGGUGUUUAUCUTT-3′) and siRNA3 (5′-GGUGUCAUCAAGGUGUUCATT-3′) were designed to target different coding regions of the human CFL1 messenger (m)RNA sequence (Gene ID, 1072).

U251 cells were seeded onto six-well plates in DMEM containing 10% fetal calf serum without penicillin or streptomycin and then incubated overnight. Cells were then transfected with CFL1-siRNAs or pcDNA3.1-CFL1 using Lipofectamin™ 2000 (Invitrogen Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s instructions. Following 6 h of transfection, the medium was replaced with complete medium and the transfection efficiency was evaluated using a fluorescence microscope (Axiovert 40 CFL; Carl Zeiss, Oberkochen, Germany). CFL1 expression was analyzed at 24, 48 and 72 h following transfection. Cells transfected with pcDNA3.1-CFL1 were selected for stable clones using DMEM containing 400 μg/ml G418 (Sigma-Aldrich).

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Manufacturer protocol

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