siRNA / miRNA gene silencing Rat - B35 cIAP2/BIRC3

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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Stealth siRNA(r)_BIRC3

Thermo Fisher Scientific

Upstream tips	Protocol tips	Downstream tips
	Transfection of siRNA was done as described previously [24]. Commercially available small interfering RNA (siRNA) duplexes directed against rat cIAP1 and cIAP2 were purchased from Invitrogen (Carlsbad, California) as described [25], and are all selected and validated Stealth siRNAs. Control siRNA duplexes were purchased from Dharmacon (Lafayette, CO). Lamin A/C siRNA (Dharmacon) was used to optimize the transfection conditions and efficiency. Additional controls of nonspecific siRNA duplex or vehicle alone were evaluated and found not to alter cIAP-1 and cIAP-2 mRNA and protein expression at the concentration of siRNA found to reduce the specific target message and protein optimally.

Protocol tips
Transfection of siRNA was done as described previously [24]. Commercially available small interfering RNA (siRNA) duplexes directed against rat cIAP1 and cIAP2 were purchased from Invitrogen (Carlsbad, California) as described [25], and are all selected and validated Stealth siRNAs. Control siRNA duplexes were purchased from Dharmacon (Lafayette, CO). Lamin A/C siRNA (Dharmacon) was used to optimize the transfection conditions and efficiency. Additional controls of nonspecific siRNA duplex or vehicle alone were evaluated and found not to alter cIAP-1 and cIAP-2 mRNA and protein expression at the concentration of siRNA found to reduce the specific target message and protein optimally.

Protocol tips

Transfection of siRNA was done as described previously [24]. Commercially available small interfering RNA (siRNA) duplexes directed against rat cIAP1 and cIAP2 were purchased from Invitrogen (Carlsbad, California) as described [25], and are all selected and validated Stealth siRNAs. Control siRNA duplexes were purchased from Dharmacon (Lafayette, CO). Lamin A/C siRNA (Dharmacon) was used to optimize the transfection conditions and efficiency. Additional controls of nonspecific siRNA duplex or vehicle alone were evaluated and found not to alter cIAP-1 and cIAP-2 mRNA and protein expression at the concentration of siRNA found to reduce the specific target message and protein optimally.

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