siRNA / miRNA gene silencing Mouse - P19 Oct4

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

Oct-3/4 siRNA (m)

Santa Cruz Biotechnology

Upstream tips	Protocol tips	Downstream tips
	The adenovirus-based vector AdFoxm1siRNA that expresses Foxm1-specific siRNA was constructed with Invitrogen Block-it Adenoviral RNAi Expression System (Cat. No K4941-00 and V492-20), following the manufacturer’s instructions. A siRNA sequence 5′-GGA CCA CTT CCC TTA CTT T-3 from mouse Foxm1 cDNA was used to design a double-strand DNA that was recombinated into the adenovirus vector. The constructions of human FOXM1-expression adenovirus AdFOXM1 and control virus AdLacZ or AdGFP were described previously (34). The large-scale adenovirus purification and viral infections were carried out as previously described (35). For siRNA treatment, mOct4 siRNA (sc-36124) and control siRNA (sc-37007) were purchased from Santa Cruz. The siRNA transfection was performed according to the manufacturer’s instructions.

Protocol tips
The adenovirus-based vector AdFoxm1siRNA that expresses Foxm1-specific siRNA was constructed with Invitrogen Block-it Adenoviral RNAi Expression System (Cat. No K4941-00 and V492-20), following the manufacturer’s instructions. A siRNA sequence 5′-GGA CCA CTT CCC TTA CTT T-3 from mouse Foxm1 cDNA was used to design a double-strand DNA that was recombinated into the adenovirus vector. The constructions of human FOXM1-expression adenovirus AdFOXM1 and control virus AdLacZ or AdGFP were described previously (34). The large-scale adenovirus purification and viral infections were carried out as previously described (35). For siRNA treatment, mOct4 siRNA (sc-36124) and control siRNA (sc-37007) were purchased from Santa Cruz. The siRNA transfection was performed according to the manufacturer’s instructions.

Protocol tips

The adenovirus-based vector AdFoxm1siRNA that expresses Foxm1-specific siRNA was constructed with Invitrogen Block-it Adenoviral RNAi Expression System (Cat. No K4941-00 and V492-20), following the manufacturer’s instructions. A siRNA sequence 5′-GGA CCA CTT CCC TTA CTT T-3 from mouse Foxm1 cDNA was used to design a double-strand DNA that was recombinated into the adenovirus vector. The constructions of human FOXM1-expression adenovirus AdFOXM1 and control virus AdLacZ or AdGFP were described previously (34). The large-scale adenovirus purification and viral infections were carried out as previously described (35). For siRNA treatment, mOct4 siRNA (sc-36124) and control siRNA (sc-37007) were purchased from Santa Cruz. The siRNA transfection was performed according to the manufacturer’s instructions.

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