siRNA / miRNA gene silencing Mouse - MC3T3-E1 SphK1

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 Sphk1 (20698) siRNA - SMARTpool

Horizon Discovery Ltd.

Upstream tips	Protocol tips	Downstream tips
	The ON-TARGET plus SMART-pool of double-stranded small interfering RNA directed against mouse SphK1 was from Dharmacon Thermo Scientific. Spns2 transient interference was achieved by double-stranded mouse specific small interfering RNAs 5′- GCUAGUAGGUAUUGGUGAA (dT)(dT) -3′ (siSpns2a), 5′-CUCAUCUUUGGAGCCAUUA (dT)(dT) -3′ (siSpns2b), and 5′-CCAUCUUCAUCUGCCUCAU (dT)(dT) -3′ (siSpns2c) from Sigma. S1P1 transient interference was achieved by double-stranded human specific small interfering RNAs 5′-GCAGCUCGGUCUCUGACUA (dT)(dT) -3′ (siS1P1a) and 5′- GGAGAACAUCUUUGUCUUG (dT)(dT) -3′ (siS1P1b) from Dharmacon Thermo Scientific. S1P3 transient interference was achieved by double-stranded human specific small interfering RNAs 5′- GAACGCAGCACUUCAGAAU (dT)(dT) -3′ (siS1P3a) and 5′- GGGAUGUGCUGGCUCAUUG (dT)(dT) -3′ (siS1P3b) from Dharmacon Thermo Scientific. Aleatory sequence scrambled siRNA was from Eurogentec (Angers, France). Transfections were carried out using the Lipofectamine 2000 reagent (Invitrogen) as previously reported (Brizuela et al., 2010).

Protocol tips
The ON-TARGET plus SMART-pool of double-stranded small interfering RNA directed against mouse SphK1 was from Dharmacon Thermo Scientific. Spns2 transient interference was achieved by double-stranded mouse specific small interfering RNAs 5′- GCUAGUAGGUAUUGGUGAA (dT)(dT) -3′ (siSpns2a), 5′-CUCAUCUUUGGAGCCAUUA (dT)(dT) -3′ (siSpns2b), and 5′-CCAUCUUCAUCUGCCUCAU (dT)(dT) -3′ (siSpns2c) from Sigma. S1P1 transient interference was achieved by double-stranded human specific small interfering RNAs 5′-GCAGCUCGGUCUCUGACUA (dT)(dT) -3′ (siS1P1a) and 5′- GGAGAACAUCUUUGUCUUG (dT)(dT) -3′ (siS1P1b) from Dharmacon Thermo Scientific. S1P3 transient interference was achieved by double-stranded human specific small interfering RNAs 5′- GAACGCAGCACUUCAGAAU (dT)(dT) -3′ (siS1P3a) and 5′- GGGAUGUGCUGGCUCAUUG (dT)(dT) -3′ (siS1P3b) from Dharmacon Thermo Scientific. Aleatory sequence scrambled siRNA was from Eurogentec (Angers, France). Transfections were carried out using the Lipofectamine 2000 reagent (Invitrogen) as previously reported (Brizuela et al., 2010).

Protocol tips

The ON-TARGET plus SMART-pool of double-stranded small interfering RNA directed against mouse SphK1 was from Dharmacon Thermo Scientific. Spns2 transient interference was achieved by double-stranded mouse specific small interfering RNAs 5′- GCUAGUAGGUAUUGGUGAA (dT)(dT) -3′ (siSpns2a), 5′-CUCAUCUUUGGAGCCAUUA (dT)(dT) -3′ (siSpns2b), and 5′-CCAUCUUCAUCUGCCUCAU (dT)(dT) -3′ (siSpns2c) from Sigma. S1P1 transient interference was achieved by double-stranded human specific small interfering RNAs 5′-GCAGCUCGGUCUCUGACUA (dT)(dT) -3′ (siS1P1a) and 5′- GGAGAACAUCUUUGUCUUG (dT)(dT) -3′ (siS1P1b) from Dharmacon Thermo Scientific. S1P3 transient interference was achieved by double-stranded human specific small interfering RNAs 5′- GAACGCAGCACUUCAGAAU (dT)(dT) -3′ (siS1P3a) and 5′- GGGAUGUGCUGGCUCAUUG (dT)(dT) -3′ (siS1P3b) from Dharmacon Thermo Scientific. Aleatory sequence scrambled siRNA was from Eurogentec (Angers, France). Transfections were carried out using the Lipofectamine 2000 reagent (Invitrogen) as previously reported (Brizuela et al., 2010).

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