siRNA / miRNA gene silencing Human - HeLa BART/ARL2BP

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 ARL2BP siRNA

Horizon Discovery Ltd.

Upstream tips	Protocol tips	Downstream tips
	Cells were grown in six-well culture dishes to 70–90% confluence. Plasmids (2 µg of pcDNA3.1 or pcDNA3.1 carrying indicated inserts and Mito-GFP; 2∶1 ratio) were co-transfected using LipofectAMINE and Plus reagents (Invitrogen) according to the manufacturer's instructions. siRNAs (25 nM) were transfected into HeLa cells using Dharmafect transfection reagent #1 (Dharmacon, Lafayette, CO) following the company's instructions. For some experiments, transfections were performed sequentially first with plasmid DNA followed 5 hours later by transfection of the ARL2 siRNA, to minimize cell toxicity evident from co-transfections. We consistently observed at least 70% transfection efficiency, using expression of fluorescence-tagged proteins to score

Protocol tips
Cells were grown in six-well culture dishes to 70–90% confluence. Plasmids (2 µg of pcDNA3.1 or pcDNA3.1 carrying indicated inserts and Mito-GFP; 2∶1 ratio) were co-transfected using LipofectAMINE and Plus reagents (Invitrogen) according to the manufacturer's instructions. siRNAs (25 nM) were transfected into HeLa cells using Dharmafect transfection reagent #1 (Dharmacon, Lafayette, CO) following the company's instructions. For some experiments, transfections were performed sequentially first with plasmid DNA followed 5 hours later by transfection of the ARL2 siRNA, to minimize cell toxicity evident from co-transfections. We consistently observed at least 70% transfection efficiency, using expression of fluorescence-tagged proteins to score

Protocol tips

Cells were grown in six-well culture dishes to 70–90% confluence. Plasmids (2 µg of pcDNA3.1 or pcDNA3.1 carrying indicated inserts and Mito-GFP; 2∶1 ratio) were co-transfected using LipofectAMINE and Plus reagents (Invitrogen) according to the manufacturer's instructions. siRNAs (25 nM) were transfected into HeLa cells using Dharmafect transfection reagent #1 (Dharmacon, Lafayette, CO) following the company's instructions. For some experiments, transfections were performed sequentially first with plasmid DNA followed 5 hours later by transfection of the ARL2 siRNA, to minimize cell toxicity evident from co-transfections. We consistently observed at least 70% transfection efficiency, using expression of fluorescence-tagged proteins to score

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