siRNA / miRNA gene silencing Human - ES2 BRCA1

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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siGENOME Human BRCA1 (672) siRNA - SMARTpool

Horizon Discovery Ltd.

Upstream tips	Protocol tips	Downstream tips
	ES2 ovarian cancer cells were verified by short tandem repeat profiling by The Centre for Applied Genomics Genetic Analysis Facility (Toronto, ON, Canada). All cells were grown in Dulbecco’s modified Eagle’s medium/F-12 supplemented with 10% FBS, 100 U/mL penicillin, 100 mg streptomycin and fungizone (Gibco Cell Culture Products/Invitrogen). A549 cells are a commonly used cell line for studies on NF-kB signaling. Si GENOME SMARTpool BRCA1-targeting siRNA, and nontargeting siRNA were purchased from Dharmacon. Western blotting confirmed specific knockdown of BRCA1 (80%) relative to nontargeting siRNA control. To determine the impact of BRCA1 on NF-kB transcriptional activity, cells were plated at 100,000 cells/well in 24-well plates and transiently transfected with 50 pmol each of DAB2, or nontargeting siRNA 24 hours after plating using Lipofectamine LTX (Invitrogen) according to the manufacturer’s protocol.

Protocol tips
ES2 ovarian cancer cells were verified by short tandem repeat profiling by The Centre for Applied Genomics Genetic Analysis Facility (Toronto, ON, Canada). All cells were grown in Dulbecco’s modified Eagle’s medium/F-12 supplemented with 10% FBS, 100 U/mL penicillin, 100 mg streptomycin and fungizone (Gibco Cell Culture Products/Invitrogen). A549 cells are a commonly used cell line for studies on NF-kB signaling. Si GENOME SMARTpool BRCA1-targeting siRNA, and nontargeting siRNA were purchased from Dharmacon. Western blotting confirmed specific knockdown of BRCA1 (80%) relative to nontargeting siRNA control. To determine the impact of BRCA1 on NF-kB transcriptional activity, cells were plated at 100,000 cells/well in 24-well plates and transiently transfected with 50 pmol each of DAB2, or nontargeting siRNA 24 hours after plating using Lipofectamine LTX (Invitrogen) according to the manufacturer’s protocol.

Protocol tips

ES2 ovarian cancer cells were verified by short tandem repeat profiling by The Centre for Applied Genomics Genetic Analysis Facility (Toronto, ON, Canada). All cells were grown in Dulbecco’s modified Eagle’s medium/F-12 supplemented with 10% FBS, 100 U/mL penicillin, 100 mg streptomycin and fungizone (Gibco Cell Culture Products/Invitrogen). A549 cells are a commonly used cell line for studies on NF-kB signaling. Si GENOME SMARTpool BRCA1-targeting siRNA, and nontargeting siRNA were purchased from Dharmacon. Western blotting confirmed specific knockdown of BRCA1 (80%) relative to nontargeting siRNA control. To determine the impact of BRCA1 on NF-kB transcriptional activity, cells were plated at 100,000 cells/well in 24-well plates and transiently transfected with 50 pmol each of DAB2, or nontargeting siRNA 24 hours after plating using Lipofectamine LTX (Invitrogen) according to the manufacturer’s protocol.

Manufacturer protocol Publication protocol 1 Relevant paper

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