siRNA / miRNA gene silencing Human - MCF-7 PRC (PGC-1α–related coactivator)/PPRC1

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 PPRC1 siRNA

Horizon Discovery Ltd.

Protocol tips
MCF-7 cells were obtained from the ATCC (Manassas, VA). ZR75.1 and TOV112D cells were a kind gift from Almac Diagnostics. Cells were all maintained in Dulbecco's modified Eagle's medium (Biowhittaker, Verviers, Belgium) supplemented with 10% (v/v) heat-inactivated FBS, 10 mM L-glutamine, and 5 mg/ml penicillin/streptomycin (complete medium) at 37 °C in a humidified 95% air, 5% CO2 atmosphere.To suppress PRC expression, siRNA pools targeting human PRC (L-016647-00-0005) was obtained from Dharmacon. Silencer negative control 1 was purchased from Thermo Fisher. Reverse siRNA transfections were carried out using Lipofectamine RNAiMAX transfection reagent (Invitrogen) according to the instructions of the manufacturer.
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