siRNA / miRNA gene silencing Human - MDA-MB-231 RAD51

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 RAD51 (5888) siRNA - SMARTpool

Horizon Discovery Ltd.

Upstream tips	Protocol tips	Downstream tips
	MDA-MB-231 human breast cancer cells (ATCC, Rockville, MD, USA) were maintained in Dulbecco’s high glucose (25 mM glucose) modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (Gibco, Carlsbad, CA, USA), 100 U/mL penicillin, and 100 μg/mL streptomycin. All cells were cultured at 37 °C in a humidified incubator with 5% CO2. MDA-MB-231 cells were seeded in six-well plates and transfected at 60% confluence with RAD51-targeting siRNA duplexes or a negative control siRNA (L-003530-00-0005; UAUCAUCGCCCAUGCAUCA, CUAAUCAGGUGGUAGCUCA, GCAGUGAUGUCCUGGAUAA, and CCAACGAUGUGAAGAAAUU) purchased from Dharmacon (Lafayette, CO, USA). For transfection, 5 μL of siRNA targeting human RAD51 (CR536559) and 5 μL of Lipofectamine were each diluted in 95 μL of reduced serum medium (Opti-MEM, Invitrogen, Carlsbad, CA, USA). The mixtures were incubated for 15 min before being added dropwise to the culture wells containing 800 μL of Opti-MEM to achieve a final siRNA concentration of 50 nM.

Protocol tips
MDA-MB-231 human breast cancer cells (ATCC, Rockville, MD, USA) were maintained in Dulbecco’s high glucose (25 mM glucose) modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (Gibco, Carlsbad, CA, USA), 100 U/mL penicillin, and 100 μg/mL streptomycin. All cells were cultured at 37 °C in a humidified incubator with 5% CO2. MDA-MB-231 cells were seeded in six-well plates and transfected at 60% confluence with RAD51-targeting siRNA duplexes or a negative control siRNA (L-003530-00-0005; UAUCAUCGCCCAUGCAUCA, CUAAUCAGGUGGUAGCUCA, GCAGUGAUGUCCUGGAUAA, and CCAACGAUGUGAAGAAAUU) purchased from Dharmacon (Lafayette, CO, USA). For transfection, 5 μL of siRNA targeting human RAD51 (CR536559) and 5 μL of Lipofectamine were each diluted in 95 μL of reduced serum medium (Opti-MEM, Invitrogen, Carlsbad, CA, USA). The mixtures were incubated for 15 min before being added dropwise to the culture wells containing 800 μL of Opti-MEM to achieve a final siRNA concentration of 50 nM.

Protocol tips

MDA-MB-231 human breast cancer cells (ATCC, Rockville, MD, USA) were maintained in Dulbecco’s high glucose (25 mM glucose) modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (Gibco, Carlsbad, CA, USA), 100 U/mL penicillin, and 100 μg/mL streptomycin. All cells were cultured at 37 °C in a humidified incubator with 5% CO2. MDA-MB-231 cells were seeded in six-well plates and transfected at 60% confluence with RAD51-targeting siRNA duplexes or a negative control siRNA (L-003530-00-0005; UAUCAUCGCCCAUGCAUCA, CUAAUCAGGUGGUAGCUCA, GCAGUGAUGUCCUGGAUAA, and CCAACGAUGUGAAGAAAUU) purchased from Dharmacon (Lafayette, CO, USA). For transfection, 5 μL of siRNA targeting human RAD51 (CR536559) and 5 μL of Lipofectamine were each diluted in 95 μL of reduced serum medium (Opti-MEM, Invitrogen, Carlsbad, CA, USA). The mixtures were incubated for 15 min before being added dropwise to the culture wells containing 800 μL of Opti-MEM to achieve a final siRNA concentration of 50 nM.

Manufacturer protocol Publication protocol 1 Relevant paper

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