Lipofectamine® 2000 Transfection Reagent

Protocol tips

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	Add diluted siRNA to diluted Lipofectamine Reagent Incubate for 5 min at RT and add to cells. Incubate cells for 1–3 days at 37°C.

Protocol tips
Add diluted siRNA to diluted Lipofectamine Reagent Incubate for 5 min at RT and add to cells. Incubate cells for 1–3 days at 37°C.

Publication protocol

For the miRNA construct, one target sequence (5′-GCAGGTCATAGTTTTGGCCACTG-3′) was selected corresponding to the open reading frame of the human HIF-1α gene (NM-001530). The construct containing a scrambled sequence (5′-CGTGGAGACGTTTTGGCCACTGA-3′) (Scrambled) was also included as a negative control; it has no significant homology with human gene sequences. They were synthesized by Invitrogen and inserted into pcDNA6.2-GW/EmGFP eukaryotic expression vectors to construct miRNA or negative control vectors, which were termed HIF-1α-miRNA and Scrambled, respectively. For gene transfection, 2×105 cells per well were set into 6-well plates and grown overnight until they were 50–80% confluent. Plasmids HIF-1α-miRNA and Scrambled were transfected into A549 cells by Lipofectamine 2000 reagent (Invitrogen) as per the manufacturer’s instructions. Cells were subcultured at a 1:5 dilution in 300 mg/ml G418-containing medium. Positive stable transfections were selected and expanded for further study. The pCLEN plasmid encoding full-length survivin was a kind gift from Dr Feng Qian (Department of Pharmacology, University of Illinois, Chicago, IL, USA). Cells were transfected twice with 2 μg of expression vector or empty pCRII-TOPO control (Invitrogen) 6 and 24 h after HIF-1α-miRNA transfection (described above) using the FuGENE 6 Transfection Reagent (Roche Diagnostics) as per the manufacturer’s recommendations. Cells were harvested 24 h after transfection for western blotting.

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Manufacturer protocol

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