siRNA / miRNA gene silencing Human - BEAS-2B RAB7A

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

Rab 7 siRNA (h)

Santa Cruz Biotechnology

Upstream tips	Protocol tips	Downstream tips
	Human bronchial epithelial cells (BEAS-2B; ATCC CRL-9609) were routinely cultured in RPMI base medium supplemented with 10% fetal bovine serum (FBS) and 1× penicillin-streptomycin in 75-cm2 tissue culture flasks at 37°C in the presence of 5% CO2. Cells were grown to approximately 75% confluence and passaged. Cells were discarded after their 9th passage. Cells were plated in a 24-well plate for all assays at a density of 250,000 cells per well. Cells were allowed to grow for approximately 48 h prior to challenge with a medium exchange at 24 h prior to challenge. Serum-starved cells were given RPMI medium lacking FBS 24 h prior to challenge. Transient expression of mCherry fusion proteins occurred through a standardized lipofection-based protocol described by Clark et al. (56). Transient silencing of a specified gene occurred through Lipofectamine LTX with Plus solution via the manufacturer's protocol. Specifically, liposomes were prepared using 1 to 5 μl siRNA (Santa Cruz), 5 μl Plus solution per well, and 5 μl Lipofectamine LTX per well

Protocol tips
Human bronchial epithelial cells (BEAS-2B; ATCC CRL-9609) were routinely cultured in RPMI base medium supplemented with 10% fetal bovine serum (FBS) and 1× penicillin-streptomycin in 75-cm2 tissue culture flasks at 37°C in the presence of 5% CO2. Cells were grown to approximately 75% confluence and passaged. Cells were discarded after their 9th passage. Cells were plated in a 24-well plate for all assays at a density of 250,000 cells per well. Cells were allowed to grow for approximately 48 h prior to challenge with a medium exchange at 24 h prior to challenge. Serum-starved cells were given RPMI medium lacking FBS 24 h prior to challenge. Transient expression of mCherry fusion proteins occurred through a standardized lipofection-based protocol described by Clark et al. (56). Transient silencing of a specified gene occurred through Lipofectamine LTX with Plus solution via the manufacturer's protocol. Specifically, liposomes were prepared using 1 to 5 μl siRNA (Santa Cruz), 5 μl Plus solution per well, and 5 μl Lipofectamine LTX per well

Protocol tips

Human bronchial epithelial cells (BEAS-2B; ATCC CRL-9609) were routinely cultured in RPMI base medium supplemented with 10% fetal bovine serum (FBS) and 1× penicillin-streptomycin in 75-cm2 tissue culture flasks at 37°C in the presence of 5% CO2. Cells were grown to approximately 75% confluence and passaged. Cells were discarded after their 9th passage. Cells were plated in a 24-well plate for all assays at a density of 250,000 cells per well. Cells were allowed to grow for approximately 48 h prior to challenge with a medium exchange at 24 h prior to challenge. Serum-starved cells were given RPMI medium lacking FBS 24 h prior to challenge. Transient expression of mCherry fusion proteins occurred through a standardized lipofection-based protocol described by Clark et al. (56). Transient silencing of a specified gene occurred through Lipofectamine LTX with Plus solution via the manufacturer's protocol. Specifically, liposomes were prepared using 1 to 5 μl siRNA (Santa Cruz), 5 μl Plus solution per well, and 5 μl Lipofectamine LTX per well

Manufacturer protocol Publication protocol 1 Relevant paper

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