siRNA / miRNA gene silencing Rat - PC12 Lsm1

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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Protocol tips
Cells were transfected with 1 µg of plasmid DNA in 12-well tissue culture plate format. Plasmids that expressed short hairpin RNA (shRNA) against specific transcripts were similarly transfected by using 1 µg DNA and induced with 300 ng/ml doxycycline. On-Target Plus SmartPool small interfering RNAs (siRNAs) against Dcp1a and Lsm1 were obtained from Dharmacon (Lafayette, CO, USA). Tet-stable PC12 cells were transfected with inducible plasmids and induced with 500 ng/ml doxycycline medium for 24 or 48 h. PC12 cells were transfected by using LipofectamineLTX (Thermo Fisher Scientific). All transfections were performed according to the manufacturer’s protocol.
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