siRNA / miRNA gene silencing Mouse - MS1 Nostrin

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® siRNA(m) Nostrin

Thermo Fisher Scientific

Upstream tips	Protocol tips	Downstream tips
	Transfection was performed using Lipofectamine LTX and Plus reagent (Invitrogen). Based on the experiment, various constructs, such as pCAG-NOSTRIN expression vector, pCAG-NOSTRINΔSH3, pCAGNOSTRINΔ(SH3 + ID), pCAG-NOSTRINΔ (SH3 + ID + HR1) or pCAG-TRAF6 were used. Control cells were transfected with empty vector backbone without dsRed. Transfected cells were incubated for 48 h and were subjected to RNA or protein isolation. In each experiment with NOSTRIN over-expression, mRNA levels were assessed by real time PCR. NOSTRIN expression levels were found to be more or less consistent between replicates as well as between experiments. Cells were also treated with NωNitro-L-arginine (L-NNA, Sigma Aldrich) at a final concentration of 100 µM for inhibition of NOS. All functional assays were performed at least 24 h after transfection and within 48 h to ensure high levels of NOSTRIN expression. For siRNA treatment cells were transfected using Lipofectamine RNAiMAX (Invitrogen). Two pre-validated Silencer Select siRNAs targeting the coding region of NOSTRIN (Assay Id: s116394 and s116396) were used for down-regulation. Control cells were treated with scrambled siRNA. Cells were treated with siRNAs in a dose dependant manner and down regulation were quantified by Real time PCR. The concentration at which maximum down regulation occurred was selected for further experiments.

Protocol tips
Transfection was performed using Lipofectamine LTX and Plus reagent (Invitrogen). Based on the experiment, various constructs, such as pCAG-NOSTRIN expression vector, pCAG-NOSTRINΔSH3, pCAGNOSTRINΔ(SH3 + ID), pCAG-NOSTRINΔ (SH3 + ID + HR1) or pCAG-TRAF6 were used. Control cells were transfected with empty vector backbone without dsRed. Transfected cells were incubated for 48 h and were subjected to RNA or protein isolation. In each experiment with NOSTRIN over-expression, mRNA levels were assessed by real time PCR. NOSTRIN expression levels were found to be more or less consistent between replicates as well as between experiments. Cells were also treated with NωNitro-L-arginine (L-NNA, Sigma Aldrich) at a final concentration of 100 µM for inhibition of NOS. All functional assays were performed at least 24 h after transfection and within 48 h to ensure high levels of NOSTRIN expression. For siRNA treatment cells were transfected using Lipofectamine RNAiMAX (Invitrogen). Two pre-validated Silencer Select siRNAs targeting the coding region of NOSTRIN (Assay Id: s116394 and s116396) were used for down-regulation. Control cells were treated with scrambled siRNA. Cells were treated with siRNAs in a dose dependant manner and down regulation were quantified by Real time PCR. The concentration at which maximum down regulation occurred was selected for further experiments.

Protocol tips

Transfection was performed using Lipofectamine LTX and Plus reagent (Invitrogen). Based on the experiment, various constructs, such as pCAG-NOSTRIN expression vector, pCAG-NOSTRINΔSH3, pCAGNOSTRINΔ(SH3 + ID), pCAG-NOSTRINΔ (SH3 + ID + HR1) or pCAG-TRAF6 were used. Control cells were transfected with empty vector backbone without dsRed. Transfected cells were incubated for 48 h and were subjected to RNA or protein isolation. In each experiment with NOSTRIN over-expression, mRNA levels were assessed by real time PCR. NOSTRIN expression levels were found to be more or less consistent between replicates as well as between experiments. Cells were also treated with NωNitro-L-arginine (L-NNA, Sigma Aldrich) at a final concentration of 100 µM for inhibition of NOS. All functional assays were performed at least 24 h after transfection and within 48 h to ensure high levels of NOSTRIN expression. For siRNA treatment cells were transfected using Lipofectamine RNAiMAX (Invitrogen). Two pre-validated Silencer Select siRNAs targeting the coding region of NOSTRIN (Assay Id: s116394 and s116396) were used for down-regulation. Control cells were treated with scrambled siRNA. Cells were treated with siRNAs in a dose dependant manner and down regulation were quantified by Real time PCR. The concentration at which maximum down regulation occurred was selected for further experiments.

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