Transient transfection of oligonucleotides

H1299-IL24^wt (1 X 10⁵) cells were seeded in six-well plates and were transiently transfected with the miR-222-3p inhibitor and miR negative control (100 nM; Dharmacon) using DOTAP:Cholesterol liposome as previously described [37, 40]. After six hours of transfection, tissue culture medium was aspirated and replenished with fresh medium. The cells were then either not treated or treated with doxycycline (1 μg/ml). Untransfected cells served as controls. The cells were harvested at 24 h after transfection. Cell lysates were prepared and used for miRNA and protein expression analysis.

H1299-IL24^wt (1 X 10⁵) cells were seeded in six-well plates and were either treated or not treated with doxycycline (1 μg/ml, Sigma Chemicals). After six hours, the medium was removed and cells were transiently transfected with miR-222-3p mimic and miR negative control (50 nM, Dharmacon) using DOTAP:Cholesterol liposome as previously described [37, 40]. Cells that were not transfected served as controls. The cells were harvested 24 h after transfection. Cell lysates were prepared and used for miRNA and protein expression analysis. Negative controls used in our miR222-3p overexpression/downregulation studies, are chemically synthesized single stranded modified RNAs. These negative controls have no homology to any known mammalian gene. Use of non-targeting inhibitors and mimics in miRNA inhibition and mimic experiments respectively will distinguish between specific inhibitor/mimic activity and their background effects. Neg.con. - denotes Negative control.