siRNA / miRNA gene silencing Mouse - 3T3-L1 ANT2

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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ANT2 siRNA (m)

Santa Cruz Biotechnology

Upstream tips	Protocol tips	Downstream tips
	3T3-L1 preadipocytes were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), at 10% CO2 and 37°C. Differentiation of 3T3-L1 cells was induced as described previously 48. Briefly, confluent cells were incubated for 2 days in a medium comprising DMEM supplemented with 10% FBS, 0.5 mM 3-isobutyl-1-methylxanthine, 1 μM dexamethasone, and 10 μg of insulin/ml. Thereafter, medium was replaced every other day with DMEM containing 10% FBS and 10 μg/ml insulin.The negative control (sc-37007) and specific siRNAs against ANT2 (sc-72506), and HIF-1α (sc-35562) were purchased from Santa Cruz Biotechnology, Inc. Transfections were performed with DharmaFECT 1 transfection reagent (Dharmacon) and conducted according to the manufacturer’s instruction

Protocol tips
3T3-L1 preadipocytes were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), at 10% CO2 and 37°C. Differentiation of 3T3-L1 cells was induced as described previously 48. Briefly, confluent cells were incubated for 2 days in a medium comprising DMEM supplemented with 10% FBS, 0.5 mM 3-isobutyl-1-methylxanthine, 1 μM dexamethasone, and 10 μg of insulin/ml. Thereafter, medium was replaced every other day with DMEM containing 10% FBS and 10 μg/ml insulin.The negative control (sc-37007) and specific siRNAs against ANT2 (sc-72506), and HIF-1α (sc-35562) were purchased from Santa Cruz Biotechnology, Inc. Transfections were performed with DharmaFECT 1 transfection reagent (Dharmacon) and conducted according to the manufacturer’s instruction

Protocol tips

3T3-L1 preadipocytes were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), at 10% CO2 and 37°C. Differentiation of 3T3-L1 cells was induced as described previously 48. Briefly, confluent cells were incubated for 2 days in a medium comprising DMEM supplemented with 10% FBS, 0.5 mM 3-isobutyl-1-methylxanthine, 1 μM dexamethasone, and 10 μg of insulin/ml. Thereafter, medium was replaced every other day with DMEM containing 10% FBS and 10 μg/ml insulin.The negative control (sc-37007) and specific siRNAs against ANT2 (sc-72506), and HIF-1α (sc-35562) were purchased from Santa Cruz Biotechnology, Inc. Transfections were performed with DharmaFECT 1 transfection reagent (Dharmacon) and conducted according to the manufacturer’s instruction

Manufacturer protocol Publication protocol 1 Relevant paper

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