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Method of Using LIC™

Preservation of LIC™ Vial
  • Preserve the lyophilized LIC™ vial at 4°C.

Preparation and storage of LIC™ stock solution
  • Add 1.85 mL of RNase-free H2O to the vial containing freeze-dried LIC™, and shake the vial gently by hand until the LIC™ is completely dissolved.
  • Let the reconstituted LIC™ stand at room temperature for 15 min.

  • The reconstituted LIC™ stock solution contains 16 mg lipid/mL in a volume of 2 mL. The stock solution must be stored at 4°C.
  • It can be stored at 4°C. for up to 1 month.

Preparation of LIC™ working solution
  • The LIC™ working solution is prepared by diluting the LIC™ stock solution with saline or serum-free culture medium.
  • Do NOT store the LIC™ working solution overnight.

Preparation of RNA/LIC™ Complex
  • A complex shall be prepared per test. The prepared complex shall be consumed during the day of preparation.
  • The maximum concentration of the prepared RNA/LIC™ complex is 50 µg/ml (RNA concentration) and the minimum amount of drop is 500 µl.
  • The compound ratio of RNA and LIC™ is 1: 16 (weight). When diluting LIC™ crude liquid as LIC™ solution, diluting in a saline solution or serum-less culture medium.
  • The RNA solution and LIC™ solution are mixed at a ratio of 1:1 (volume).

  • Slowly add the RNA solution to an equal volume of LIC™ working solution. Gently mix the RNA/ LIC™ solution by hand for 5 seconds.
  • Further dilute the RNA/ LIC™ solution with RNase-free saline or serum-free culture medium as necessary.
    Recommendation: Use sterile polystyrene tubes to form the RNA/ LIC™ complex.

Addition of Cell Culture Medium and RNA/LIC™ Complex
  • Spread the cells in the cultivation plate and culture them for a night. required cell density to be a 50~60% confluence the next day
  • After the completion of change of culture medium the next day, add the prepared RNA/LIC™ complex solution to the culture medium.
    The RNA/LIC™ complex solution shall be at the amount of 1/9 of culture medium.

Precautions
  • All procedures must be carried out under sterile conditions.
  • The RNA/LIC solution described in this protocol is for IN VITRO experiments only. Do NOT use this RNA/LIC solution for in vivo experiments. For in vivo experiments, a specific formulation of the RNA/LIC complex is needed.

A sample preparation of RNA/LIC complex
  • Preparation of 1 µM siRNA/LIC solution using a 21-base-pair siRNA (double-stranded RNA, molecular weight 13,400).

  • Prepare 2 µM siRNA solution (A) and LIC™ working solution (B) as follows.
    (A): 2 µM siRNA solution (500 µL)
       20 µM siRNA solution 50 µL(13.4 µg RNA)
       saline or serum-free culture medium 450 µL
    (B): LIC working solution (500 µL)
       LIC stock solution (16 mg lipid/mL) 13.4 µL (214.4 µg lipid)
       saline or serum-free culture medium 486.6 µL
  • Slowly add solution (A) to an equal volume of solution (B). Gently mix the RNA/LIC solution by hand for 5 seconds.
  • Further dilute the RNA/LIC solution with saline or serum-free culture medium as necessary. Change the culture medium of the cells. Add the RNA/LIC solution to the cells in a volume equal to 1/9 the volume of the culture medium.

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