Usinglipofectamine rnaimax transfection reagent

T-REx-HRE
cells were seeded
at 20 000 cells/well on 6-well plates and incubated for 24
h such that cell density reached 50–70% confluence just prior
to transfection with siRNA. Cells were transfected with siRNA using
Lipofectamine RNAiMAX transfection reagent (Life Technologies) according
to the manufacturer’s instructions for “forward transfection”.
Briefly, cell culture media was removed from cells and replaced with
serum-free OptiMEM cell culture medium (Life Technologies). siRNA
and Lipofectamine were separately, diluted in a volume of OptiMEM
equivalent to 10% of the final volume of cells, and incubated at RT
for 5 min. The diluted oligonucleotides and transfection reagent were
then combined, mixed gently, and incubated at RT for 10 min. The siRNA-Lipofectamine
complexes were added dropwise to cells which were then incubated at
37 °C for 24 h. The final concentration of siRNA was 5 nM and
the final amount of Lipofectamine was 0.2% v/v for all experiments.
Cells were either transfected with EPAS1 (HIF-2α) siRNA (Silencer
Select predesigned annealed human oligonucleotide duplex, s4700, Life
Technologies), scrambled siRNA (Silencer Select negative control number
2, Life Technologies) or vehicle alone. Following transfection, cells
were incubated in hypoxic or aerobic conditions for 24 h in the presence
or absence of 1 μg/mL for 24 h, then harvested for total RNA
extraction.