siRNA / miRNA gene silencing Mouse - CT26 caspase-8

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 Casp8 (12370) siRNA - SMARTpool

Horizon Discovery Ltd.

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	All cell lines were purchased from American Type Culture Collection (Manassas, VA, USA). Mouse colon adenocarcinoma CT26 cell lines were cultured in RPMI 1640 (Gibco) supplemented with 10% heat-inactivated FBS (PAA), 2 mM GlutaMAX I CTS (Gibco) and 100 U/mL penicillin + 100 µg/mL streptomycin (Gibco). A total of 1 × 105 CT26 cells per well were plated in six-well plates and allowed to reach 50% confluence on the day of transfection. siRNA specific for mouse caspase-8 (ON-TARGET plus Mouse Casp8 siRNA SMART pool L-043044-00-0010) and an unrelated control (ON-TARGET plus Non-targeting pool D-001810-10-05) were purchased from Dharmacon. Cells were transfected with siRNA at a final concentration of 25 nM using Lipofectamine® RNAiMAX Transfection Reagent (Invitrogen) according to the manufacturer's instructions. Knockdown of caspase-8 was confirmed by western blotting.

Protocol tips
All cell lines were purchased from American Type Culture Collection (Manassas, VA, USA). Mouse colon adenocarcinoma CT26 cell lines were cultured in RPMI 1640 (Gibco) supplemented with 10% heat-inactivated FBS (PAA), 2 mM GlutaMAX I CTS (Gibco) and 100 U/mL penicillin + 100 µg/mL streptomycin (Gibco). A total of 1 × 105 CT26 cells per well were plated in six-well plates and allowed to reach 50% confluence on the day of transfection. siRNA specific for mouse caspase-8 (ON-TARGET plus Mouse Casp8 siRNA SMART pool L-043044-00-0010) and an unrelated control (ON-TARGET plus Non-targeting pool D-001810-10-05) were purchased from Dharmacon. Cells were transfected with siRNA at a final concentration of 25 nM using Lipofectamine® RNAiMAX Transfection Reagent (Invitrogen) according to the manufacturer's instructions. Knockdown of caspase-8 was confirmed by western blotting.

Protocol tips

All cell lines were purchased from American Type Culture Collection (Manassas, VA, USA). Mouse colon adenocarcinoma CT26 cell lines were cultured in RPMI 1640 (Gibco) supplemented with 10% heat-inactivated FBS (PAA), 2 mM GlutaMAX I CTS (Gibco) and 100 U/mL penicillin + 100 µg/mL streptomycin (Gibco). A total of 1 × 105 CT26 cells per well were plated in six-well plates and allowed to reach 50% confluence on the day of transfection. siRNA specific for mouse caspase-8 (ON-TARGET plus Mouse Casp8 siRNA SMART pool L-043044-00-0010) and an unrelated control (ON-TARGET plus Non-targeting pool D-001810-10-05) were purchased from Dharmacon. Cells were transfected with siRNA at a final concentration of 25 nM using Lipofectamine® RNAiMAX Transfection Reagent (Invitrogen) according to the manufacturer's instructions. Knockdown of caspase-8 was confirmed by western blotting.

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