siRNA / miRNA gene silencing Human - SHSY5Y ATG16L1

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 human ATG16L1 siRNA

Horizon Discovery Ltd.

Upstream tips	Protocol tips	Downstream tips
	Human neuroblastoma cells from the SH-SY5Y cell line (CRL2266) were purchased from ATCCCells were maintained in Dulbecco’s Modified Eagle’s Medium/Nutrient Mixture F-12 (DMEM/F12; Thermo Fisher Scientific, 11320–033), to which 10% fetal bovine serum (Hyclone, SH30370.03) and antibiotics (100 U/ml penicillin, 100 μg/ml streptomycin; Thermo Fisher Scientific, 15140122) had been added. Cells were grown at 37°C in an atmosphere of 5% CO2. ON-TARGETplus human ATG16L1 siRNA (L-021033) or Non-targeting siRNA (D-001810) were obtained from Dharmacon. SH-SY5Y cells grown in 6-well plates were transfected with siRNA, at a concentration of 30 pmol/well with Lipofectamine RNAiMAX (Thermo Fisher Scientific, 13778) twice according to the manufacturer’s instructions. 72 h after transfection, cells were used for further analyses.

Protocol tips
Human neuroblastoma cells from the SH-SY5Y cell line (CRL2266) were purchased from ATCCCells were maintained in Dulbecco’s Modified Eagle’s Medium/Nutrient Mixture F-12 (DMEM/F12; Thermo Fisher Scientific, 11320–033), to which 10% fetal bovine serum (Hyclone, SH30370.03) and antibiotics (100 U/ml penicillin, 100 μg/ml streptomycin; Thermo Fisher Scientific, 15140122) had been added. Cells were grown at 37°C in an atmosphere of 5% CO2. ON-TARGETplus human ATG16L1 siRNA (L-021033) or Non-targeting siRNA (D-001810) were obtained from Dharmacon. SH-SY5Y cells grown in 6-well plates were transfected with siRNA, at a concentration of 30 pmol/well with Lipofectamine RNAiMAX (Thermo Fisher Scientific, 13778) twice according to the manufacturer’s instructions. 72 h after transfection, cells were used for further analyses.

Protocol tips

Human neuroblastoma cells from the SH-SY5Y cell line (CRL2266) were purchased from ATCCCells were maintained in Dulbecco’s Modified Eagle’s Medium/Nutrient Mixture F-12 (DMEM/F12; Thermo Fisher Scientific, 11320–033), to which 10% fetal bovine serum (Hyclone, SH30370.03) and antibiotics (100 U/ml penicillin, 100 μg/ml streptomycin; Thermo Fisher Scientific, 15140122) had been added. Cells were grown at 37°C in an atmosphere of 5% CO2. ON-TARGETplus human ATG16L1 siRNA (L-021033) or Non-targeting siRNA (D-001810) were obtained from Dharmacon. SH-SY5Y cells grown in 6-well plates were transfected with siRNA, at a concentration of 30 pmol/well with Lipofectamine RNAiMAX (Thermo Fisher Scientific, 13778) twice according to the manufacturer’s instructions. 72 h after transfection, cells were used for further analyses.

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