siRNA / miRNA gene silencing Human - THP-1 NOTCH1

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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Stealth siRNA™ NOTCH1

Thermo Fisher Scientific

Upstream tips	Protocol tips	Downstream tips
	The THP-1 (acute monoblastic leukemia) cell line was supplied by the Health Science Research Resources Bank (Osaka, Japan),The cells were cultured in 10% fetal calf serum (FCS)-supplemented RPMI-1640 medium at 37˚C in a humidified 5% CO2 atmosphere .pre-designed siRNAs (Stealth siRNA™)targeting NOTCH1 (siN1: HSS107248, 107249, and 181550) was purchased from Life Technologies (Carlsbad, CA, USA). The stealth RNAi negative control Duplex was used as a control siRNA. Each siRNA was transfected at a concentration of 40 nM using the pipette tip chamber-based electroporation, the Neon™ Transfection System (Life Technologies), according to the manufacturer’s instructions. After transfection, the cells were quickly transferred into culture medium.

Protocol tips
The THP-1 (acute monoblastic leukemia) cell line was supplied by the Health Science Research Resources Bank (Osaka, Japan),The cells were cultured in 10% fetal calf serum (FCS)-supplemented RPMI-1640 medium at 37˚C in a humidified 5% CO2 atmosphere .pre-designed siRNAs (Stealth siRNA™)targeting NOTCH1 (siN1: HSS107248, 107249, and 181550) was purchased from Life Technologies (Carlsbad, CA, USA). The stealth RNAi negative control Duplex was used as a control siRNA. Each siRNA was transfected at a concentration of 40 nM using the pipette tip chamber-based electroporation, the Neon™ Transfection System (Life Technologies), according to the manufacturer’s instructions. After transfection, the cells were quickly transferred into culture medium.

Protocol tips

The THP-1 (acute monoblastic leukemia) cell line was supplied by the Health Science Research Resources Bank (Osaka, Japan),The cells were cultured in 10% fetal calf serum (FCS)-supplemented RPMI-1640 medium at 37˚C in a humidified 5% CO2 atmosphere .pre-designed siRNAs (Stealth siRNA™)targeting NOTCH1 (siN1: HSS107248, 107249, and 181550) was purchased from Life Technologies (Carlsbad, CA, USA). The stealth RNAi negative control Duplex was used as a control siRNA. Each siRNA was transfected at a concentration of 40 nM using the pipette tip chamber-based electroporation, the Neon™ Transfection System (Life Technologies), according to the manufacturer’s instructions. After transfection, the cells were quickly transferred into culture medium.

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