siRNA / miRNA gene silencing Mouse - MS1 Cdh5

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 Mouse Cdh5 (12562) siRNA - SMARTpool

Horizon Discovery Ltd.

Upstream tips	Protocol tips	Downstream tips
	HUVECs and MS-1 cells were incubated with 40 nM ON-TARGETplus SMARTpool duplex siRNA targeting Cdh5 (Dharmacon, #L003641; L-041968) or 20 nM siRNA targeting S1pr1 (Dharmacon, # D-001810) using 2 μl/ml Lipofectamine 2000 (Invitrogen, #11668027) or Lipofectamine RNAiMAX (Invitrogen, #13778075) in OptiMEM (GIBCO) as previously described (Rudini et al., 2008). Cells were cultured in complete medium for a further 48 h (S1pr1 siRNA) or 78 h (Cdh5 siRNA) before starvation (18 h for MS1, 6 h for HUVECs), and subsequent stimulations with VEGFR2 and S1PR1 agonists (hVEGF165, SEW2871, S1P from Cayman Chemicals) and processing for staining. siCONTROL Non-Targeting siRNA Pool (Dharmacon, #D-001810) at the same concentration was used as a control for all the siRNA interference assay.

Protocol tips
HUVECs and MS-1 cells were incubated with 40 nM ON-TARGETplus SMARTpool duplex siRNA targeting Cdh5 (Dharmacon, #L003641; L-041968) or 20 nM siRNA targeting S1pr1 (Dharmacon, # D-001810) using 2 μl/ml Lipofectamine 2000 (Invitrogen, #11668027) or Lipofectamine RNAiMAX (Invitrogen, #13778075) in OptiMEM (GIBCO) as previously described (Rudini et al., 2008). Cells were cultured in complete medium for a further 48 h (S1pr1 siRNA) or 78 h (Cdh5 siRNA) before starvation (18 h for MS1, 6 h for HUVECs), and subsequent stimulations with VEGFR2 and S1PR1 agonists (hVEGF165, SEW2871, S1P from Cayman Chemicals) and processing for staining. siCONTROL Non-Targeting siRNA Pool (Dharmacon, #D-001810) at the same concentration was used as a control for all the siRNA interference assay.

Protocol tips

HUVECs and MS-1 cells were incubated with 40 nM ON-TARGETplus SMARTpool duplex siRNA targeting Cdh5 (Dharmacon, #L003641; L-041968) or 20 nM siRNA targeting S1pr1 (Dharmacon, # D-001810) using 2 μl/ml Lipofectamine 2000 (Invitrogen, #11668027) or Lipofectamine RNAiMAX (Invitrogen, #13778075) in OptiMEM (GIBCO) as previously described (Rudini et al., 2008). Cells were cultured in complete medium for a further 48 h (S1pr1 siRNA) or 78 h (Cdh5 siRNA) before starvation (18 h for MS1, 6 h for HUVECs), and subsequent stimulations with VEGFR2 and S1PR1 agonists (hVEGF165, SEW2871, S1P from Cayman Chemicals) and processing for staining. siCONTROL Non-Targeting siRNA Pool (Dharmacon, #D-001810) at the same concentration was used as a control for all the siRNA interference assay.

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