siRNA / miRNA gene silencing Human - HCT-116 PTBP1

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

Stealth Select_PTBP1 siRNA

Thermo Fisher Scientific

Upstream tips	Protocol tips	Downstream tips
	The human colon cancer cell lines HCT116 was obtained from the American Type Culture Collection in 2001. Stocks were prepared after passage 2 and stored in liquid nitrogen. All experiments were performed with cells of passage of <8. These cell line was authenticated by morphologic inspection, short tandem repeat profiling, and Mycoplasma testing by the ATCC. Mycoplasma testing was done also by the authors. Cells were grown in Dulbecco's modified Eagle medium supplemented with 10% fetal bovine serum at 37°C in a humidified incubator with 5% CO2.The small interfering RNA (siRNA) for PTBP1 (Stealth Select RNAi, HSS143520) and negative control siRNA (Negative Control Hi GC) were purchased from Invitrogen. Cells were transfected with siRNA in 20 nmol/L concentration using Lipofectamine RNAiMAX (Invitrogen) according to the manufacturer's protocols.

Protocol tips
The human colon cancer cell lines HCT116 was obtained from the American Type Culture Collection in 2001. Stocks were prepared after passage 2 and stored in liquid nitrogen. All experiments were performed with cells of passage of <8. These cell line was authenticated by morphologic inspection, short tandem repeat profiling, and Mycoplasma testing by the ATCC. Mycoplasma testing was done also by the authors. Cells were grown in Dulbecco's modified Eagle medium supplemented with 10% fetal bovine serum at 37°C in a humidified incubator with 5% CO2.The small interfering RNA (siRNA) for PTBP1 (Stealth Select RNAi, HSS143520) and negative control siRNA (Negative Control Hi GC) were purchased from Invitrogen. Cells were transfected with siRNA in 20 nmol/L concentration using Lipofectamine RNAiMAX (Invitrogen) according to the manufacturer's protocols.

Protocol tips

The human colon cancer cell lines HCT116 was obtained from the American Type Culture Collection in 2001. Stocks were prepared after passage 2 and stored in liquid nitrogen. All experiments were performed with cells of passage of <8. These cell line was authenticated by morphologic inspection, short tandem repeat profiling, and Mycoplasma testing by the ATCC. Mycoplasma testing was done also by the authors. Cells were grown in Dulbecco's modified Eagle medium supplemented with 10% fetal bovine serum at 37°C in a humidified incubator with 5% CO2.The small interfering RNA (siRNA) for PTBP1 (Stealth Select RNAi, HSS143520) and negative control siRNA (Negative Control Hi GC) were purchased from Invitrogen. Cells were transfected with siRNA in 20 nmol/L concentration using Lipofectamine RNAiMAX (Invitrogen) according to the manufacturer's protocols.

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