siRNA / miRNA gene silencing Human - A498 PIK3CA

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 PIK3CA (5290) siRNA - SMARTpool

Horizon Discovery Ltd.

Upstream tips	Protocol tips	Downstream tips
	A498 cell lines were acquired from the American Type Culture Collection (Manassas, VA, USA). Cells undergo annual STR analysis for genetic confirmation. All cell lines were cultured in Dulbecco’s Modified Eagle Medium (DMEM, Gibco/Life Technologies, Carlsbad, CA, USA) supplemented with 10% FBS (Gemini Bio-Products, West Sacramento, CA, USA), non-essential amino acids, L-glutamine, penicillin, and streptomycin. All cultures were maintained at 37° C in 5% CO2. Cells were transfected with human SMART pool ON-TARGETplus PIK3CA (L-003018-00-0005), and ON-TARGETplus Non-targeting pool (D-001810-10-05) siRNAs siRNAs using DharmaFECT 1 transfection reagent (T-2-001-02) (GE Dharmacon) according to the manufacturer’s protocol. Transfections were performed in serum-reduced, antibiotic-free DharmaFECT Cell Culture Reagent (DCCR) (B-004500-100) (GE Dharmacon).

Protocol tips
A498 cell lines were acquired from the American Type Culture Collection (Manassas, VA, USA). Cells undergo annual STR analysis for genetic confirmation. All cell lines were cultured in Dulbecco’s Modified Eagle Medium (DMEM, Gibco/Life Technologies, Carlsbad, CA, USA) supplemented with 10% FBS (Gemini Bio-Products, West Sacramento, CA, USA), non-essential amino acids, L-glutamine, penicillin, and streptomycin. All cultures were maintained at 37° C in 5% CO2. Cells were transfected with human SMART pool ON-TARGETplus PIK3CA (L-003018-00-0005), and ON-TARGETplus Non-targeting pool (D-001810-10-05) siRNAs siRNAs using DharmaFECT 1 transfection reagent (T-2-001-02) (GE Dharmacon) according to the manufacturer’s protocol. Transfections were performed in serum-reduced, antibiotic-free DharmaFECT Cell Culture Reagent (DCCR) (B-004500-100) (GE Dharmacon).

Protocol tips

A498 cell lines were acquired from the American Type Culture Collection (Manassas, VA, USA). Cells undergo annual STR analysis for genetic confirmation. All cell lines were cultured in Dulbecco’s Modified Eagle Medium (DMEM, Gibco/Life Technologies, Carlsbad, CA, USA) supplemented with 10% FBS (Gemini Bio-Products, West Sacramento, CA, USA), non-essential amino acids, L-glutamine, penicillin, and streptomycin. All cultures were maintained at 37° C in 5% CO2. Cells were transfected with human SMART pool ON-TARGETplus PIK3CA (L-003018-00-0005), and ON-TARGETplus Non-targeting pool (D-001810-10-05) siRNAs siRNAs using DharmaFECT 1 transfection reagent (T-2-001-02) (GE Dharmacon) according to the manufacturer’s protocol. Transfections were performed in serum-reduced, antibiotic-free DharmaFECT Cell Culture Reagent (DCCR) (B-004500-100) (GE Dharmacon).

Manufacturer protocol Publication protocol 1 Relevant paper

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