siRNA / RNAi /miRNA transfection Rat - IEC-6 Cationic lipid based

RNAi or RNA interference is a common method to suppress gene expression in vitro/in vivo by utilizing the inherent microRNA machinery, without introducing a total gene knockout. 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.

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Found 1 matching solution for this experiment

siRNA Transfection Reagent

Santa Cruz Biotechnology

Upstream tips	Protocol tips	Downstream tips
Cells (2×105 per well) were seeded in a 6-well tissue culture plate in 2 ml antibiotic-free normal growth medium supplemented with FBS and incubated at 37°C in a CO2 incubator until the cells were 60–80% confluent. This took 18–24 h.	For each transfection, 0.8 ml siRNA transfection medium was added to each tube containing the siRNA transfection reagent mixture (solution A + solution B). After incubating for 6 h at 37°C in a CO2 incubator, the transfection mixture was removed and replaced with 1X normal growth medium for an additional 24 h, and the cells were subjected to western blot analysis of β-arrestin-2.

Upstream tips
Cells (2×105 per well) were seeded in a 6-well tissue culture plate in 2 ml antibiotic-free normal growth medium supplemented with FBS and incubated at 37°C in a CO2 incubator until the cells were 60–80% confluent. This took 18–24 h.
Protocol tips
For each transfection, 0.8 ml siRNA transfection medium was added to each tube containing the siRNA transfection reagent mixture (solution A + solution B). After incubating for 6 h at 37°C in a CO2 incubator, the transfection mixture was removed and replaced with 1X normal growth medium for an additional 24 h, and the cells were subjected to western blot analysis of β-arrestin-2.

Manufacturer protocol Publication protocol 1 Relevant paper

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