RNA Oligonucleotides - miRNA Transfection

MicroRNAs (miRNAs) are a class of non-coding RNAs that are crucial in gene expression regulation; they are involved in processes such as stem cell function, cancer development and viral infections. miRNA encoding genes initially produce primary miRNA, which will undergo processing and be exported out of the nucleus, resulting in the formation of mature miRNA. Mature miRNA associates with Argonaute protein to form the RNA-induced silencing complex (RISC). 

See below for a general protocol and recommendations. 

Materials

  1. Tissue culture plate (6-well, 12-well, 24-well, or 96-well)
  2. Growth medium
  3. Fetal Bovine Serum (FBS)
  4. Liposomal Transfection Reagent
  5. Vials/tubes
  6. 37°C incubator

Protocol

Cell preparation
  1. Seed cells 18-24 hours before transfection at a density of 1-3 x 105 cells per well in 2.0 ml of growth medium, with antibiotics and serum if normally required. 
  2. Incubate the cells at 37°C until approximately 70-90% confluency. 
  3. Suspension cells: plate 3-5 x 105 cells in 0.8 ml of serum free medium without antibiotics.  
Transfection
  1. Prepare the complexes in a sterile micro-centrifuge for each transfection sample as follows: 
    • Solution A: dilute 5-100 nM synthetic miRNA into 125 µl of serum-free, antibiotic-free medium. 
    • Solution B: Mix the Transfection Reagent thoroughly before use. Then dilute 4-10 µl of reagent in 125 µl serum-free, antibiotic-free medium.  
  2. Incubate Solution A and Solution B separately at room temperature for 5 minutes.
  3. Combine the solutions by mixing Solution A and Solution B thoroughly to create a RNA/oligo-liposome solution. Then, incubate for 20 minutes at room temperature. This will allow RNA/oligo-liposome complexes to form.  
  4. Adherent cells: add 0.8 ml of serum-free antibiotic-free medium to the RNA/oligo-liposome complexes, mix gently. Remove the growth medium from the adherent cells and add 1.0 ml of RNA/oligo-liposome solution to each well containing cells. Incubate at 37°C for 5-8 hours to allow RNA/oligo-liposome complexes to form. 
    Suspension cells: add 0.2 ml of RNA/oligo-liposome solution into each well containing the suspension cells in the serum and antibiotic-free medium. Incubate the plate at 37°C for 5-8 hours to allow RNA/oligo-liposome complexes to form.  
  5. Remove the RNA/oligo-liposome solution from all the wells. Add either 2.0 ml of growth medium containing serum and antibiotics or 0.1 ml of FBS into each well. Incubate the cells at 37°C for 6-72 hours. Optimal incubation time varies.
  6. After 6-72 hours, monitor and assay gene expression.