siRNA / miRNA gene silencing Rat - F98 Faslg

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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Silencer®_Faslg siRNA (r)

Thermo Fisher Scientific

Upstream tips	Protocol tips	Downstream tips
	Predesigned siRNAs directed against rat FasL mRNA (GenBank: NM_012908) were purchased from Ambion (Austin, Texas; siRNA IDs 197271, 48716, and 197272). SiRNAs were transiently transfected into wild-type 9L and F98 glioma cells and screened by immunoblotting for optimal knock-down 48 hours after transfection. A scramble siRNA was used as negative control (Ambion product # AM4636). Transient transfections were performed using 100 nM siRNA combined with siPORT lipid transfection reagent (Ambion, Inc.) in optiMEM media (Gibco) according to the manufacturers' recommendations. Briefly, glioma cells were plated in 100-mm-diameter tissue culture dishes at 5 × 105 cells/plate in medium supplemented with 10% FBS. The following day medium was removed and replaced with medium containing 0.1% FBS. Approximately 1 hour later, siRNA:lipid complexes were added to the cells and allowed to incubate for 48 hours before cells were harvested as described above.

Protocol tips
Predesigned siRNAs directed against rat FasL mRNA (GenBank: NM_012908) were purchased from Ambion (Austin, Texas; siRNA IDs 197271, 48716, and 197272). SiRNAs were transiently transfected into wild-type 9L and F98 glioma cells and screened by immunoblotting for optimal knock-down 48 hours after transfection. A scramble siRNA was used as negative control (Ambion product # AM4636). Transient transfections were performed using 100 nM siRNA combined with siPORT lipid transfection reagent (Ambion, Inc.) in optiMEM media (Gibco) according to the manufacturers' recommendations. Briefly, glioma cells were plated in 100-mm-diameter tissue culture dishes at 5 × 105 cells/plate in medium supplemented with 10% FBS. The following day medium was removed and replaced with medium containing 0.1% FBS. Approximately 1 hour later, siRNA:lipid complexes were added to the cells and allowed to incubate for 48 hours before cells were harvested as described above.

Protocol tips

Predesigned siRNAs directed against rat FasL mRNA (GenBank: NM_012908) were purchased from Ambion (Austin, Texas; siRNA IDs 197271, 48716, and 197272). SiRNAs were transiently transfected into wild-type 9L and F98 glioma cells and screened by immunoblotting for optimal knock-down 48 hours after transfection. A scramble siRNA was used as negative control (Ambion product # AM4636). Transient transfections were performed using 100 nM siRNA combined with siPORT lipid transfection reagent (Ambion, Inc.) in optiMEM media (Gibco) according to the manufacturers' recommendations. Briefly, glioma cells were plated in 100-mm-diameter tissue culture dishes at 5 × 105 cells/plate in medium supplemented with 10% FBS. The following day medium was removed and replaced with medium containing 0.1% FBS. Approximately 1 hour later, siRNA:lipid complexes were added to the cells and allowed to incubate for 48 hours before cells were harvested as described above.

Manufacturer protocol Publication protocol 1 Relevant paper

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