siRNA / miRNA gene silencing Human - Caco-2 PAK1

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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Protocol tips
"Caco-2 cells were treated with small interfering RNA (siRNA)using a modification of our previously published methods (Forsyth
et al., 2010). Briefly, 105 cells are combined with 11 picomoles of siRNA in 50 ll Lipofectamine (Invitrogen) and 50 ul Optimem (Invitrogen), mixed by gently shaking and then plated on Type 1 collagencoated Corning 12 mm PET culture inserts (#3460) with addition of another 400 ul (upper well) and 1 ml (lower well) Optimem. Inserts are used for permeability studies 72 to 96 hours after plating, when
the cells are confluent. PAK1 and iNOS siRNA were On Target Smartpool from Dharmacon (Lafayette, CO), whereas control (nontargeting) siRNA was from Santa Cruz Biotechnology (Santa
Cruz, CA)"
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