siRNA / miRNA gene silencing Human - TF‐1 IFI16

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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Found 1 matching solution for this experiment

Hs_IFI16_1 FlexiTube siRNA

Qiagen

Upstream tips	Protocol tips	Downstream tips
	Human CD34+ TF-1 cells, was grown in RPMI1640 media, and HeLa cells in D-MEM media, all supplemented with 10% heatinactivated foetal bovine serum (FBS). TF-1 cells were grown in the presence of 2ng/ml recombinant human GM-CSF (R&D Systems, Abingdon, UK).Transient transfections were performed using the Amaxa Nucleofector system (program T-003). For IFI16 knockdown studies, 0.5–2μg of IFI16 siRNA (code: SI00445676; Qiagen, Crawley, UK) or control siRNA (code: 1027280) were co-transfected with pcDNA4-N1ICD or empty vector. Efficiency of siRNA knockdown was assessed by FACS analysis and RT-PCR.

Protocol tips
Human CD34+ TF-1 cells, was grown in RPMI1640 media, and HeLa cells in D-MEM media, all supplemented with 10% heatinactivated foetal bovine serum (FBS). TF-1 cells were grown in the presence of 2ng/ml recombinant human GM-CSF (R&D Systems, Abingdon, UK).Transient transfections were performed using the Amaxa Nucleofector system (program T-003). For IFI16 knockdown studies, 0.5–2μg of IFI16 siRNA (code: SI00445676; Qiagen, Crawley, UK) or control siRNA (code: 1027280) were co-transfected with pcDNA4-N1ICD or empty vector. Efficiency of siRNA knockdown was assessed by FACS analysis and RT-PCR.

Protocol tips

Human CD34+ TF-1 cells, was grown in RPMI1640 media, and HeLa cells in D-MEM media, all supplemented with 10% heatinactivated foetal bovine serum (FBS). TF-1 cells were grown in the presence of 2ng/ml recombinant human GM-CSF (R&D Systems, Abingdon, UK).Transient transfections were performed using the Amaxa Nucleofector system (program T-003). For IFI16 knockdown studies, 0.5–2μg of IFI16 siRNA (code: SI00445676; Qiagen, Crawley, UK) or control siRNA (code: 1027280) were co-transfected with pcDNA4-N1ICD or empty vector. Efficiency of siRNA knockdown was assessed by FACS analysis and RT-PCR.

Manufacturer protocol Publication protocol 1 Relevant paper

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