siRNA / miRNA gene silencing Rat - B35 Alkbh1

miRNA is the inherent gene silencing machinery which can have more than one mRNA target, whereas siRNA can be designed to target a particular mRNA target. By design, both siRNA and miRNA are 20-25 nucleotides in length. The target sequence for siRNAs is usually located within the open reading frame, between 50 and 100 nucleotides downstream of the start codon. There are two ways in which cells can be transfected with desired RNAi: 1. Direct transfection (with calcium phosphate co-precipitation or cationic lipid mediated transfection using lipofectamine or oligofectamine), and 2. Making RNAi lentiviral constructs (followed by transformation and transduction). Lentiviral constructs are time consuming, but provide a more permanent expression of RNAi in the cells, and consistent gene silencing. Direct transfection of oligonucleotides provides temporary genetic suppression. Traditional methods like calcium phosphate co-precipitation have challenges like low efficiency, poor reproducibility and cell toxicity. Whereas, cationic lipid-based transfection reagents are able to overcome these challenges, along with applicability to a large variety of eukaryotic cell lines. When using oligos, the ideal concentration lies between 10-50nM for effective transfection.

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Silencer® Select_Alkbh1 siRNA (r)

Thermo Fisher Scientific

Upstream tips	Protocol tips	Downstream 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.

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.

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.

Manufacturer protocol Publication protocol 1 Relevant paper

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