Silencer® Select_Alkbh1 siRNA (r)

Protocol tips

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	Cells were plated at a density of 2 × 105 per well in 6-well plates or 4 × 104 in 24 well plates. The next day medium was changed to high glucose DMEM without FBS or antibiotics and the cells were transfected with Alkbh1 siRNA or Negative control siRNA using Lipofectamine RNAimax according to the manufacturer’s protocol. After 48 hours the medium was changed to full GM and the cells allowed to recover for 24 hours before stress experiments or sample collection for RNA or western blotting analysis. Transfection efficiency was > 50% as evaluated using western blotting (Figure 1C). I/R stress was performed as described previously, however, due to Lipofectamine induced toxicity to the cells, the optimal drug induced oxidative stresses were evaluated to be 400µM arsenite and 150µg/ml antimycin-A for 4 hours after several trials.

Protocol tips

Cells were plated at a density of 2 × 105 per well in 6-well plates or 4 × 104 in 24 well plates. The next day medium was changed to high glucose DMEM without FBS or antibiotics and the cells were transfected with Alkbh1 siRNA or Negative control siRNA using Lipofectamine RNAimax according to the manufacturer’s protocol. After 48 hours the medium was changed to full GM and the cells allowed to recover for 24 hours before stress experiments or sample collection for RNA or western blotting analysis. Transfection efficiency was > 50% as evaluated using western blotting (Figure 1C). I/R stress was performed as described previously, however, due to Lipofectamine induced toxicity to the cells, the optimal drug induced oxidative stresses were evaluated to be 400µM arsenite and 150µg/ml antimycin-A for 4 hours after several trials.

Publication protocol

Cells were plated at a density of 2 × 105 per well in 6-well plates or 4 × 104 in 24 well plates. The next day medium was changed to high glucose DMEM without FBS or antibiotics and the cells were transfected with Alkbh1 siRNA or Negative control siRNA using Lipofectamine RNAimax according to the manufacturer’s protocol. After 48 hours the medium was changed to full GM and the cells allowed to recover for 24 hours before stress experiments or sample collection for RNA or western blotting analysis. Transfection efficiency was > 50% as evaluated using western blotting (Figure 1C). I/R stress was performed as described previously, however, due to Lipofectamine induced toxicity to the cells, the optimal drug induced oxidative stresses were evaluated to be 400µM arsenite and 150µg/ml antimycin-A for 4 hours after several trials.

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Manufacturer protocol

Download the product protocol from Thermo Fisher Scientific for Silencer® Select_Alkbh1 siRNA (r) below.

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