siRNA / miRNA gene silencing Rat - RBL-2H3 Vamp7

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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Found 1 matching solution for this experiment

Silencer® Select_Vamp7 siRNA(r)

Thermo Fisher Scientific

Upstream tips	Protocol tips	Downstream tips
	Amaxa nucleofection: RBL‐2H3 cells were resuspended at 1e6 cells/100 μl in Amaxa nucleofection solution L (Amaxa) [26] and transferred to electroporation cuvettes, and siRNA was added at various concentrations, and cells were electroporated using Amaxa program L‐029. Five hundred microlitres of prewarmed culture media was added to each cuvette, and cells were then added to 1.5‐ml prewarmed culture media in 6‐well plates. Cells were incubated for 24–48 h, and cells were assessed for transfection efficiency [using 100 nm siGLO siRNA or 2 μg of a green fluorescent protein (GFP)‐expressing plasmid; pmaxGFP (Amaxa)] by fluorescence microscopy or by using flow‐cytometry on FACScan (BD Biosciences, Franklin Lakes, NJ, USA) using a FITC filter. For siRNA knock‐down experiments, cells were incubated for 24–48 h and then washed and lysed for RNA isolation or SDS‐PAGE/immunoblotting or transferred to 24‐well plates for degranulation assays.

Protocol tips
Amaxa nucleofection: RBL‐2H3 cells were resuspended at 1e6 cells/100 μl in Amaxa nucleofection solution L (Amaxa) [26] and transferred to electroporation cuvettes, and siRNA was added at various concentrations, and cells were electroporated using Amaxa program L‐029. Five hundred microlitres of prewarmed culture media was added to each cuvette, and cells were then added to 1.5‐ml prewarmed culture media in 6‐well plates. Cells were incubated for 24–48 h, and cells were assessed for transfection efficiency [using 100 nm siGLO siRNA or 2 μg of a green fluorescent protein (GFP)‐expressing plasmid; pmaxGFP (Amaxa)] by fluorescence microscopy or by using flow‐cytometry on FACScan (BD Biosciences, Franklin Lakes, NJ, USA) using a FITC filter. For siRNA knock‐down experiments, cells were incubated for 24–48 h and then washed and lysed for RNA isolation or SDS‐PAGE/immunoblotting or transferred to 24‐well plates for degranulation assays.

Protocol tips

Amaxa nucleofection: RBL‐2H3 cells were resuspended at 1e6 cells/100 μl in Amaxa nucleofection solution L (Amaxa) [26] and transferred to electroporation cuvettes, and siRNA was added at various concentrations, and cells were electroporated using Amaxa program L‐029. Five hundred microlitres of prewarmed culture media was added to each cuvette, and cells were then added to 1.5‐ml prewarmed culture media in 6‐well plates. Cells were incubated for 24–48 h, and cells were assessed for transfection efficiency [using 100 nm siGLO siRNA or 2 μg of a green fluorescent protein (GFP)‐expressing plasmid; pmaxGFP (Amaxa)] by fluorescence microscopy or by using flow‐cytometry on FACScan (BD Biosciences, Franklin Lakes, NJ, USA) using a FITC filter. For siRNA knock‐down experiments, cells were incubated for 24–48 h and then washed and lysed for RNA isolation or SDS‐PAGE/immunoblotting or transferred to 24‐well plates for degranulation assays.

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