A 50 nM of small interfering RNA (siRNA) oligonucleotides against PPARγ (sc-29455, Santa Cruz Biotechnology) were transfected into Caco-2 cells in a 12 well plate (1.5 X 105 cells/well) by using Lipofactamine RNAimax transfection reagent (Invitrogen). Control cells were transfected with Allstars negative siRNA control (Qiagen). The knockdown efficiency of PPARγ siRNA was confirmed by western blotting.
Lipofactamine rnaimax transfection reagent
Manufactured by Thermo Fisher Scientific
Lipofectamine RNAiMAX is a lipid-based transfection reagent used for the delivery of small interfering RNA (siRNA) and other RNA molecules into mammalian cells. It facilitates the efficient uptake and intracellular delivery of RNA for gene silencing applications.
Automatically generated - may contain errors
2 protocols using lipofactamine rnaimax transfection reagent
1
Knockdown of PPARγ in Caco-2 Cells
2
siRNA Knockdown Optimization Protocol
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The nucleolin targeting siRNA was obtained from Qiagen (SI02654925). LRRC59 targeting siRNA was described previously [27] (link). Scrambled control siRNA was obtained from Thermo Scientific Dharmacon (CD-001810-01-20). For siRNA transfection studies, U2OSR1 cells (1x105cells/ml) were seeded out and after 24 h the cells were transfected with 50 nM of the nucleolin targeting siRNA and control targeting siRNA, and 75 nM of the LRRC59 targeting siRNA using Lipofactamine RNAiMax Transfection Reagent (Invitrogen) according to the procedure given by the company. Seven hours after transfection, 10% FBS was added to the cells, and the cells were cultured for 72 h before further experiments.
Transient expression of myc-FGF1 was performed by transfecting HEK 293 cells with plasmid DNA using Escort IV Transfection Reagent (Sigma) in Minimum Essential Medium (MEM, Gibco) according to the manufacturer’s protocol. After 7 h the medium was changed for DMEM supplemented with 10 % FBS and the cells were grown for 24 h.
Transient expression of myc-FGF1 was performed by transfecting HEK 293 cells with plasmid DNA using Escort IV Transfection Reagent (Sigma) in Minimum Essential Medium (MEM, Gibco) according to the manufacturer’s protocol. After 7 h the medium was changed for DMEM supplemented with 10 % FBS and the cells were grown for 24 h.
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