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Transfection of siRNA and plasmid

YS Yuntong Sun
YS Yuntao Sun
SC Shenyuan Chen
YY Yifan Yu
YM Yongjun Ma
FS Fengtian Sun
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Specific targeting siRNA and overexpressing plasmid were designed and synthesized by Genepharma (Suzhou, China). The full-length cDNA of the human HIF-1α gene was inserted into the pCDH-CMV-MCS-GFP + Puro plasmid vector to construct the overexpression plasmid. Cells were resuspended and seeded in 6-well plates (2 × 105 cells/well). When the cell density reached 50–70%, cells were transfected with siRNA and plasmid using Lipofectamine 2000 (Life Technologies, USA) in serum-free medium according to the manufacturer’s instructions. At 6 h after transfection, cells were exposed to the fresh complete medium and cultured for another 24 h. The sequences of siRNA were as follows:

siRNA NC: sense: UUCUCCGAACGUGUCACGUTT.

siRNA NC: antisense: ACGUGACACGUUCGGAGAATT.

si-HIF-1α: sense: CUCCCUAUAUCCCAAUGGATT.

si-HIF-1α: antisense: UCCAUUGGGAUAUAGGGAGTT.

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