siRNA / miRNA gene silencing Human - HEK293 TRIM43

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

siGENOME TRIM43 siRNA

Horizon Discovery Ltd.

Protocol tips
HEK 293 cells (all purchased from ATCC) as well as Huh7 (provided by M. Farzan, Scripps Institute Florida), were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, Thermo Fisher Scientific) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS; Thermo Fisher Scientific), 2 mM GlutaMAX (Thermo Fisher Scientific), and 1% (v/v) penicillin-streptomycin (Thermo Fisher Scientific) under standard culture conditions.HEK 293T cells were seeded into 24- or 12-well plates (~ 2.5 or 5 × 105 cells per well). The next day, cells were transfected with 60 nM or 120 nM of SiRNAs targeting TRIM43 (siGENOME SMARTpool M-007127-01-0050), using Lipofectamine RNAiMAX (Thermo Fisher Scientific) according to the manufacturer’s instructions. At 48 h post-transfection, knockdown efficiency of endogenous TRIM proteins was determined by qRT-PCR or immunoblot (IB) analysis, as described below.
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