Silencer® siRNA(m) Nostrin

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	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.

Publication protocol

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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