siRNA / miRNA gene silencing Mouse - MC3T3-E1 Fdps

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 Fdps (110196) siRNA - Individual,

Horizon Discovery Ltd.

Upstream tips	Protocol tips	Downstream tips
	Four pure individual ON-TARGETplus siRNAs all designed to target murine FPPS (target sequences: siRNA1, 50 -GUC AAG UAC AAG ACG GCUU-30 ; siRNA2, 50 -GAA AAG AGG UAC AAA UCGA-30 ; siRNA3, 50 -AGA AAG UGA CCC CGG AAUU-30 ; siRNA4, 50 -CCU AGA GUA CAA UGC CUUA-30 ) and a nontargeting control siRNA (sense, 50 -UAG CGA CUA AAC ACA UCA AUU-30 ; antisense, 50 -UUA UCG CUG AUU UGU GUA GUU-30 ) were purchased from Dharmacon (USA). DharmaFECT 4 (DF4, Dharmacon) was used as the cationic lipid cell transfection reagent in complete media. Mature osteoclasts were prepared as described above, residual monocytes were removed from culture, and siRNA transfection was immediately performed. Transfection reagent DF4 and siRNA were prepared according to the manufacturer’s instructions (Dharmacon). Final dosing concentrations of all siRNAs provided to each well were 125 nM in a total volume of 1.0 μL of DF4. Cell uptake of siRNA complexes was performed by incubating cells with siRNA complexes in complete media with 30 ng/mL M-CSF and 200 ng/mL RANKL at 37 C with 5% CO2. Nonspecific knockdown from DF4 transfection was assessed by using nontargeting siRNA dosed under identical conditions. Multiple repeated siRNA cell transfections were performed on alternating days as specified in each figure legend. MC3T3-E1 cells were cultured at 4 104 cells/well in 24-well plates in complete media overnight. Transfections were performed early the next day. Final dosing concentrations of all siRNAs provided to each well were identical to osteoclasts. Cell uptake of siRNA complexes was performed by incubating the cells with siRNA complexes in complete media with 50 μg/mL AA and 10 mM β-glycerophosphate at 37 C with 5% CO2.

Protocol tips
Four pure individual ON-TARGETplus siRNAs all designed to target murine FPPS (target sequences: siRNA1, 50 -GUC AAG UAC AAG ACG GCUU-30 ; siRNA2, 50 -GAA AAG AGG UAC AAA UCGA-30 ; siRNA3, 50 -AGA AAG UGA CCC CGG AAUU-30 ; siRNA4, 50 -CCU AGA GUA CAA UGC CUUA-30 ) and a nontargeting control siRNA (sense, 50 -UAG CGA CUA AAC ACA UCA AUU-30 ; antisense, 50 -UUA UCG CUG AUU UGU GUA GUU-30 ) were purchased from Dharmacon (USA). DharmaFECT 4 (DF4, Dharmacon) was used as the cationic lipid cell transfection reagent in complete media. Mature osteoclasts were prepared as described above, residual monocytes were removed from culture, and siRNA transfection was immediately performed. Transfection reagent DF4 and siRNA were prepared according to the manufacturer’s instructions (Dharmacon). Final dosing concentrations of all siRNAs provided to each well were 125 nM in a total volume of 1.0 μL of DF4. Cell uptake of siRNA complexes was performed by incubating cells with siRNA complexes in complete media with 30 ng/mL M-CSF and 200 ng/mL RANKL at 37 C with 5% CO2. Nonspecific knockdown from DF4 transfection was assessed by using nontargeting siRNA dosed under identical conditions. Multiple repeated siRNA cell transfections were performed on alternating days as specified in each figure legend. MC3T3-E1 cells were cultured at 4 104 cells/well in 24-well plates in complete media overnight. Transfections were performed early the next day. Final dosing concentrations of all siRNAs provided to each well were identical to osteoclasts. Cell uptake of siRNA complexes was performed by incubating the cells with siRNA complexes in complete media with 50 μg/mL AA and 10 mM β-glycerophosphate at 37 C with 5% CO2.

Protocol tips

Four pure individual ON-TARGETplus siRNAs all designed to target murine FPPS (target sequences: siRNA1, 50 -GUC AAG UAC AAG ACG GCUU-30 ; siRNA2, 50 -GAA AAG AGG UAC AAA UCGA-30 ; siRNA3, 50 -AGA AAG UGA CCC CGG AAUU-30 ; siRNA4, 50 -CCU AGA GUA CAA UGC CUUA-30 ) and a nontargeting control siRNA (sense, 50 -UAG CGA CUA AAC ACA UCA AUU-30 ; antisense, 50 -UUA UCG CUG AUU UGU GUA GUU-30 ) were purchased from Dharmacon (USA). DharmaFECT 4 (DF4, Dharmacon) was used as the cationic lipid cell transfection reagent in complete media. Mature osteoclasts were prepared as described above, residual monocytes were removed from culture, and siRNA transfection was immediately performed. Transfection reagent DF4 and siRNA were prepared according to the manufacturer’s instructions (Dharmacon). Final dosing concentrations of all siRNAs provided to each well were 125 nM in a total volume of 1.0 μL of DF4. Cell uptake of siRNA complexes was performed by incubating cells with siRNA complexes in complete media with 30 ng/mL M-CSF and 200 ng/mL RANKL at 37 C with 5% CO2. Nonspecific knockdown from DF4 transfection was assessed by using nontargeting siRNA dosed under identical conditions. Multiple repeated siRNA cell transfections were performed on alternating days as specified in each figure legend. MC3T3-E1 cells were cultured at 4 104 cells/well in 24-well plates in complete media overnight. Transfections were performed early the next day. Final dosing concentrations of all siRNAs provided to each well were identical to osteoclasts. Cell uptake of siRNA complexes was performed by incubating the cells with siRNA complexes in complete media with 50 μg/mL AA and 10 mM β-glycerophosphate at 37 C with 5% CO2.

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