siRNA / miRNA gene silencing Rat - MTLn3 Rhoa

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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ON-TARGETplus Rat Rhoa (117273) siRNA - SMARTpool

Horizon Discovery Ltd.

Upstream tips	Protocol tips	Downstream tips
	The following rat siRNA reagents from Dharmacon (Thermo Scientific, Lafayette, CO, USA) were used: RhoC (L-089673), p190RhoGAP (M-088596), p115RhoGEF (L-099751), LARG (L-101376), DLC-1 (L-088843), RhoA L-095222 siRNA. siRNA duplexes were designed against the following gene sequences: Wave2 (59-AAACCTATAACAGTGTGACG-39) and N-WASP (59-AAGACGAGATGCTCCAAATGG-39) (Sarmiento et al., 2008). siRNA were transfected into MTLn3 cells at 100 nM each using Oligofectamine (Invitrogen,Carlsbad, CA) (Bravo-Cordero et al., 2011). The transfection was terminated after 4 hours by addition of 26serum-containing medium. All experiments were performed 48 hours after transfection with more than 95% knockdown efficiency for the siRNA used (Bravo-Cordero et al., 2011). Control scrambled siRNA and cofilin siRNA (59-AAGGTGTTCAATGACATGAAA-39) sequences were described previously (Sidani et al., 2007).

Protocol tips
The following rat siRNA reagents from Dharmacon (Thermo Scientific, Lafayette, CO, USA) were used: RhoC (L-089673), p190RhoGAP (M-088596), p115RhoGEF (L-099751), LARG (L-101376), DLC-1 (L-088843), RhoA L-095222 siRNA. siRNA duplexes were designed against the following gene sequences: Wave2 (59-AAACCTATAACAGTGTGACG-39) and N-WASP (59-AAGACGAGATGCTCCAAATGG-39) (Sarmiento et al., 2008). siRNA were transfected into MTLn3 cells at 100 nM each using Oligofectamine (Invitrogen,Carlsbad, CA) (Bravo-Cordero et al., 2011). The transfection was terminated after 4 hours by addition of 26serum-containing medium. All experiments were performed 48 hours after transfection with more than 95% knockdown efficiency for the siRNA used (Bravo-Cordero et al., 2011). Control scrambled siRNA and cofilin siRNA (59-AAGGTGTTCAATGACATGAAA-39) sequences were described previously (Sidani et al., 2007).

Protocol tips

The following rat siRNA reagents from Dharmacon (Thermo Scientific, Lafayette, CO, USA) were used: RhoC (L-089673), p190RhoGAP (M-088596), p115RhoGEF (L-099751), LARG (L-101376), DLC-1 (L-088843), RhoA L-095222 siRNA. siRNA duplexes were designed against the following gene sequences: Wave2 (59-AAACCTATAACAGTGTGACG-39) and N-WASP (59-AAGACGAGATGCTCCAAATGG-39) (Sarmiento et al., 2008). siRNA were transfected into MTLn3 cells at 100 nM each using Oligofectamine (Invitrogen,Carlsbad, CA) (Bravo-Cordero et al., 2011). The transfection was terminated after 4 hours by addition of 26serum-containing medium. All experiments were performed 48 hours after transfection with more than 95% knockdown efficiency for the siRNA used (Bravo-Cordero et al., 2011). Control scrambled siRNA and cofilin siRNA (59-AAGGTGTTCAATGACATGAAA-39) sequences were described previously (Sidani et al., 2007).

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