siRNA / miRNA gene silencing Human - Keratinocytes STAT1

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 STAT1 (6772) siRNA - SMARTpool

Horizon Discovery Ltd.

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	Normal adult human keratinocytes were obtained by trypsinization of skin samples from patients undergoing plastic surgery as previously described [13]. Second-passage keratinocytes were grown in keratinocyte serum-free medium (KSFM) (Gibco, Invitrogen, Carlsbad, CA). 24 hours before stimulation with IFNα (1000 U/ml, cat. no. 11200–1, R&D Systems, Oxon, UK), the medium was changed to keratinocyte basal medium (KBM, the same as KSFM but without growth factors) in which the cells were stimulated. Cells were grown at 37˚C and 5% CO2 in an incubator. The Regional Ethical Committee of Region Midtjylland, Denmark approved the experiments with cultured human keratinocytes (M-20110027). Cultured human keratinocytes were grown to 60–70% confluency. Before transfection, the cells were changed to medium without growth factors (KBM). siRNA directed against STAT1 (cat. no. L-003543-00-0005; Dharmacon, Lafayette, CO) was preincubated with Dharmafect-2 transfection reagent (Dharmacon) in KBM for 20 minutes. The formed siRNA/transfection reagent complexes were added to the cells to a final concentration of 10 nM. As negative controls, cells were transfected with siControl nontargeting pool siRNA (cat. no. D001810-10-05, Dharmacon) or the transfection reagent alone (mock). Five hours after transfection, the medium was changed to keratinocyte growth medium (growth factors included). 24 hours before stimulation, the medium was changed to KBM

Protocol tips
Normal adult human keratinocytes were obtained by trypsinization of skin samples from patients undergoing plastic surgery as previously described [13]. Second-passage keratinocytes were grown in keratinocyte serum-free medium (KSFM) (Gibco, Invitrogen, Carlsbad, CA). 24 hours before stimulation with IFNα (1000 U/ml, cat. no. 11200–1, R&D Systems, Oxon, UK), the medium was changed to keratinocyte basal medium (KBM, the same as KSFM but without growth factors) in which the cells were stimulated. Cells were grown at 37˚C and 5% CO2 in an incubator. The Regional Ethical Committee of Region Midtjylland, Denmark approved the experiments with cultured human keratinocytes (M-20110027). Cultured human keratinocytes were grown to 60–70% confluency. Before transfection, the cells were changed to medium without growth factors (KBM). siRNA directed against STAT1 (cat. no. L-003543-00-0005; Dharmacon, Lafayette, CO) was preincubated with Dharmafect-2 transfection reagent (Dharmacon) in KBM for 20 minutes. The formed siRNA/transfection reagent complexes were added to the cells to a final concentration of 10 nM. As negative controls, cells were transfected with siControl nontargeting pool siRNA (cat. no. D001810-10-05, Dharmacon) or the transfection reagent alone (mock). Five hours after transfection, the medium was changed to keratinocyte growth medium (growth factors included). 24 hours before stimulation, the medium was changed to KBM

Protocol tips

Normal adult human keratinocytes were obtained by trypsinization of skin samples from patients undergoing plastic surgery as previously described [13]. Second-passage keratinocytes were grown in keratinocyte serum-free medium (KSFM) (Gibco, Invitrogen, Carlsbad, CA). 24 hours before stimulation with IFNα (1000 U/ml, cat. no. 11200–1, R&D Systems, Oxon, UK), the medium was changed to keratinocyte basal medium (KBM, the same as KSFM but without growth factors) in which the cells were stimulated. Cells were grown at 37˚C and 5% CO2 in an incubator. The Regional Ethical Committee of Region Midtjylland, Denmark approved the experiments with cultured human keratinocytes (M-20110027). Cultured human keratinocytes were grown to 60–70% confluency. Before transfection, the cells were changed to medium without growth factors (KBM). siRNA directed against STAT1 (cat. no. L-003543-00-0005; Dharmacon, Lafayette, CO) was preincubated with Dharmafect-2 transfection reagent (Dharmacon) in KBM for 20 minutes. The formed siRNA/transfection reagent complexes were added to the cells to a final concentration of 10 nM. As negative controls, cells were transfected with siControl nontargeting pool siRNA (cat. no. D001810-10-05, Dharmacon) or the transfection reagent alone (mock). Five hours after transfection, the medium was changed to keratinocyte growth medium (growth factors included). 24 hours before stimulation, the medium was changed to KBM

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