siRNA / miRNA gene silencing Rat - 3Y1 Acsl3

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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siRNA(r) NM_057107 Acsl3

Sigma-Aldrich

Upstream tips	Protocol tips	Downstream tips
	Rat Acsl1, Acsl3, and Acsl4 siRNAs (20 nM) (siRNA ID numbers 178 SASI_Rn01_00073026, SASI_Rn01_00100113, and SASI_Rn01_00041844; 179 Sigma) or rat cPLA2α siRNA (5 nM) (siRNA ID numbers SASI_Rn02_ 180 00267738) as well as negative control siRNA (Sigma) were transfected 181 into 3Y1 cells using Lipofectamine RNAiMAX reagent according to the 182 manufacturer's instructions. Two days after transfection, the media 183 were replaced with phenol red-free DMEM supplemented with 2% FCS. 184 After 24 h culture, 1 ng/ml IL-1β was added to the culture to activate 185 the cells. The efficacy of each siRNA was assessed by real-time PCR or 186 immunoblotting.

Protocol tips
Rat Acsl1, Acsl3, and Acsl4 siRNAs (20 nM) (siRNA ID numbers 178 SASI_Rn01_00073026, SASI_Rn01_00100113, and SASI_Rn01_00041844; 179 Sigma) or rat cPLA2α siRNA (5 nM) (siRNA ID numbers SASI_Rn02_ 180 00267738) as well as negative control siRNA (Sigma) were transfected 181 into 3Y1 cells using Lipofectamine RNAiMAX reagent according to the 182 manufacturer's instructions. Two days after transfection, the media 183 were replaced with phenol red-free DMEM supplemented with 2% FCS. 184 After 24 h culture, 1 ng/ml IL-1β was added to the culture to activate 185 the cells. The efficacy of each siRNA was assessed by real-time PCR or 186 immunoblotting.

Protocol tips

Rat Acsl1, Acsl3, and Acsl4 siRNAs (20 nM) (siRNA ID numbers 178 SASI_Rn01_00073026, SASI_Rn01_00100113, and SASI_Rn01_00041844; 179 Sigma) or rat cPLA2α siRNA (5 nM) (siRNA ID numbers SASI_Rn02_ 180 00267738) as well as negative control siRNA (Sigma) were transfected 181 into 3Y1 cells using Lipofectamine RNAiMAX reagent according to the 182 manufacturer's instructions. Two days after transfection, the media 183 were replaced with phenol red-free DMEM supplemented with 2% FCS. 184 After 24 h culture, 1 ng/ml IL-1β was added to the culture to activate 185 the cells. The efficacy of each siRNA was assessed by real-time PCR or 186 immunoblotting.

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