siRNA / miRNA gene silencing Rat - C6 (rat glioma) p53

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.

1 Matching solution
Start a discussion

Start discussion

No discussions found

Start your discussion

Share your thoughts or question with experts in your field

Start a discussion

Found 1 matching solution for this experiment

ON-TARGETplus Rat Tp53 (24842) siRNA - SMARTpool

Horizon Discovery Ltd.

Upstream tips	Protocol tips	Downstream tips
	A siRNA for p53, which targeted the RNA coding sequence, was designed by Dharmacon (ON-TARGET plus SMARTpool, Dharmacon Corporation, Lafayette, CO, USA). Negative control and GAPDH siRNAs were purchased from Ambion (Silencer Select Predesigned siRNA, Ambion, Austin, TX, USA). The siRNAs were transfected through electroporation, as specified in the instruction manual (Amaxa, Germany). After transfection, cells were cultured for 48 h to detect target expression. Briefly, 106 cells were trypsinized and resuspended in 100 μL of Nucleofector solution (Amaxa), and 100 nM of siRNA duplexes was electroporated.

Protocol tips
A siRNA for p53, which targeted the RNA coding sequence, was designed by Dharmacon (ON-TARGET plus SMARTpool, Dharmacon Corporation, Lafayette, CO, USA). Negative control and GAPDH siRNAs were purchased from Ambion (Silencer Select Predesigned siRNA, Ambion, Austin, TX, USA). The siRNAs were transfected through electroporation, as specified in the instruction manual (Amaxa, Germany). After transfection, cells were cultured for 48 h to detect target expression. Briefly, 106 cells were trypsinized and resuspended in 100 μL of Nucleofector solution (Amaxa), and 100 nM of siRNA duplexes was electroporated.

Protocol tips

A siRNA for p53, which targeted the RNA coding sequence, was designed by Dharmacon (ON-TARGET plus SMARTpool, Dharmacon Corporation, Lafayette, CO, USA). Negative control and GAPDH siRNAs were purchased from Ambion (Silencer Select Predesigned siRNA, Ambion, Austin, TX, USA). The siRNAs were transfected through electroporation, as specified in the instruction manual (Amaxa, Germany). After transfection, cells were cultured for 48 h to detect target expression. Briefly, 106 cells were trypsinized and resuspended in 100 μL of Nucleofector solution (Amaxa), and 100 nM of siRNA duplexes was electroporated.

Manufacturer protocol Publication protocol 1 Relevant paper

Can't find the product you've used to perform this experiment? It would be great if you can help us by Adding a product!

Outsource your experiment

Fill out your contact details and receive price quotes in your Inbox

Outsource experiment