siRNA / miRNA gene silencing Human - U251 Beclin 1

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

ON-TARGETplus Human BECN1 siRNA

Horizon Discovery Ltd.

Upstream tips	Protocol tips	Downstream tips
	The human glioblastoma cell line U251 was a gift from Dr. Jong Yun (Penn State College of Medicine). All cell culture media were supplemented with 10% fetal bovine serum, 100 units/ml penicillin, and 100 μg/ml streptomycin. Cells were maintained at 37 °C in a humidified atmosphere containing 5% CO2/95% air. Cells were cultured in six‐well plates at 30–40% confluence on the day of transfection. Control or Beclin 1 (Dharmacon, J‐010552‐05) siRNA at final concentration of 17 nM was mixed with 2.5 μl RNAi MAX (Invitrogen) for each well, according to the manufacturer's protocol. After 72 h of transfection, cells were collected for further experiments.

Protocol tips
The human glioblastoma cell line U251 was a gift from Dr. Jong Yun (Penn State College of Medicine). All cell culture media were supplemented with 10% fetal bovine serum, 100 units/ml penicillin, and 100 μg/ml streptomycin. Cells were maintained at 37 °C in a humidified atmosphere containing 5% CO2/95% air. Cells were cultured in six‐well plates at 30–40% confluence on the day of transfection. Control or Beclin 1 (Dharmacon, J‐010552‐05) siRNA at final concentration of 17 nM was mixed with 2.5 μl RNAi MAX (Invitrogen) for each well, according to the manufacturer's protocol. After 72 h of transfection, cells were collected for further experiments.

Protocol tips

The human glioblastoma cell line U251 was a gift from Dr. Jong Yun (Penn State College of Medicine). All cell culture media were supplemented with 10% fetal bovine serum, 100 units/ml penicillin, and 100 μg/ml streptomycin. Cells were maintained at 37 °C in a humidified atmosphere containing 5% CO2/95% air. Cells were cultured in six‐well plates at 30–40% confluence on the day of transfection. Control or Beclin 1 (Dharmacon, J‐010552‐05) siRNA at final concentration of 17 nM was mixed with 2.5 μl RNAi MAX (Invitrogen) for each well, according to the manufacturer's protocol. After 72 h of transfection, cells were collected for further experiments.

Manufacturer protocol Publication protocol 1 Relevant paper

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