siRNA / miRNA gene silencing Rat - NRVM( ATG7

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
Neonatal rats at postnatal day 0 to day 2 were sacrificed by decapitation and subsequent heart removal. Ventricular myocytes were then isolated using the Cellutron Neomyocytes isolation system (Cellutron Life Technology, Baltimore, MD) following the manufacturer's instructions [22]. Cells collected were suspended in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 µM BrdU, and 100 U/ml penicillin/streptomycin (Invitrogen, Carlsbad, CA). To selectively enrich cardiomyocytes, the cells were preplated in 100 mm non-coated dishes for 1 hour. The resulting suspended cells were counted with a hemocytometer and then plated evenly on 1% gelatin-coated plates in appropriate cell densities. The plated cells were then cultured in a 5% CO2 incubator at 37°C for at least 24 hours before the medium was changed to meet the needs of the follow-up experiments. The siRNA's specific for Atg7 (siAtg7: Cat. # SI01729119) and the siRNA targeting luciferase serving as a control siRNA (siLuc: 5′-AACGTACGCGGAATACTTCGA-3′) were all purchased from Qiagen (Valencia, CA). LipofectamineTM 2000 transfection reagent (Invitrogen) was used for siRNA transfection following the manufacturer's protocol. Transfection of cultured NRVMs with siRNA was generally started at 48–72 hours after NRVMs were plated . Six hours after the transfection, the siRNA-containing medium was replaced with the fresh medium containing 2% FBS. . For the Atg7 knockdown experiments, Atg7 siRNA were introduced along the second round of sip62 or siLuc transfection.
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