siRNA / miRNA gene silencing Mouse - 3T3-L1 BMP-3b/GDF10

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- Gdf10 siRNA

Thermo Fisher Scientific

Upstream tips	Protocol tips	Downstream tips
	3T3-L1 cells were obtained from the American Type Culture Collection and subcultured in DMEM containing 10% calf serum. 3T3-L1 cells were differentiated with the SVF as described above. Chinese hamster ovary (CHO) cells were obtained from the American Type Culture Collection and maintained in α-minimum essential medium (Invitrogen) containing 10% FBS or Opti-MEM (Invitrogen) without FBS. An siRNA specific for mouse BMP-3b (Silencer Select Pre-designed s66568) was purchased from Ambion (Austin, TX, USA). The targeted nucleotide sequence of the siRNA was CCACATGCCCTATATCCTT. Silencer Select Negative Control #1 (Ambion) was used as a control. For siRNA-mediated knockdown of 3T3-L1 cells, the cells were transfected with each siRNA (50 nm) using a Nucleofactor, plated at 1.25 × 105 cells cm−2 and then differentiated after 4 h.

Protocol tips
3T3-L1 cells were obtained from the American Type Culture Collection and subcultured in DMEM containing 10% calf serum. 3T3-L1 cells were differentiated with the SVF as described above. Chinese hamster ovary (CHO) cells were obtained from the American Type Culture Collection and maintained in α-minimum essential medium (Invitrogen) containing 10% FBS or Opti-MEM (Invitrogen) without FBS. An siRNA specific for mouse BMP-3b (Silencer Select Pre-designed s66568) was purchased from Ambion (Austin, TX, USA). The targeted nucleotide sequence of the siRNA was CCACATGCCCTATATCCTT. Silencer Select Negative Control #1 (Ambion) was used as a control. For siRNA-mediated knockdown of 3T3-L1 cells, the cells were transfected with each siRNA (50 nm) using a Nucleofactor, plated at 1.25 × 105 cells cm−2 and then differentiated after 4 h.

Protocol tips

3T3-L1 cells were obtained from the American Type Culture Collection and subcultured in DMEM containing 10% calf serum. 3T3-L1 cells were differentiated with the SVF as described above. Chinese hamster ovary (CHO) cells were obtained from the American Type Culture Collection and maintained in α-minimum essential medium (Invitrogen) containing 10% FBS or Opti-MEM (Invitrogen) without FBS. An siRNA specific for mouse BMP-3b (Silencer Select Pre-designed s66568) was purchased from Ambion (Austin, TX, USA). The targeted nucleotide sequence of the siRNA was CCACATGCCCTATATCCTT. Silencer Select Negative Control #1 (Ambion) was used as a control. For siRNA-mediated knockdown of 3T3-L1 cells, the cells were transfected with each siRNA (50 nm) using a Nucleofactor, plated at 1.25 × 105 cells cm−2 and then differentiated after 4 h.

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