siRNA / miRNA gene silencing Rat - UMR‐106 Ampkα1/Prkaa1

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.

Start discussion

No discussions found

Start your discussion

Share your thoughts or question with experts in your field

Start a discussion

Found 1 matching solution for this experiment

Protocol tips
total of 1.5X 105 cells were seeded 24 hours before the experiment in antibiotic-free medium. Cells were transfected with ONTARGETplus Rat SMARTpool Ampka1 and Ampka2 siRNA (each 50 nmol/l) (Thermo Fisher Scientific, Waltham, MA) or 100 nmol/l nonspecific siRNA (Thermo Fisher Scientific) using DharmaFECT1transfection reagent. Forty-eight hours after transfection, cells were treated with 1,25(OH)2D3 (100 nmol/l) for another 24 hours and harvested.
Can't find the product you've used to perform this experiment? It would be great if you can help us by Adding a product!

Outsource your experiment

Fill out your contact details and receive price quotes in your Inbox

  Outsource experiment
Become shareholder Discussions About us Contact Privacy Terms