RNAifectin Transfection Reagent, 1 ml RNAifectinTM is a transfection reagent specially formulated with multiple cationic polymers. It is suitable for the transfection of RNAi oligos into cultured eukaryotic cells.
Transfection protocol Use the following conditions as guidelines to transfect mammalian cells in a 6-well or 35mm dish format. For other culture vessels, please refer to Table 1.
1. Eighteen to twenty-four hours prior to transfection, seed cells at a density of 1-3 x 105 cells per well in 2.0ml of appropriate growth medium (with serum and antibiotics if normally required). Incubate the cells at 37˚C in a CO
2 incubator until cells are 70% to 90% confluent at the time of transfection.
Suspension Cells: Just prior to preparing complexes, plate 3-5 x105 cells in 0.8ml of serum-free medium without antibiotics.
Since transfection efficiency is sensitive to culture confluence, it is important to maintain a standard seeding protocol throughout all of your experiments.
2. For each transfection sample, prepare the complexes in sterile micro centrifuge tubes as follows:
Solution A: Dilute 1-3μg of RNAi oligos into 125μl of serum-free, antibiotic-free medium.
Solution B: Mix RNAifectin
TM reagent thoroughly prior to use, then dilute 4-10μl of reagent in 125μl serum-free, antibiotic-free medium.
Incubate solution A and B separately at room temperature for 5 minutes.
3. Combine solutions A and B, mix thoroughly to ensure uniform distribution and incubate for 20 minutes at room temperature to allow RNA-liposome complexes to form.
4. Adherent cells ONLY (For suspension cells, go directly to step 5b):
Add 0.8ml of serum-free, antibiotic-free medium to the RNAifectin
TM -RNA complex. Mix solution gently.
5a. Adherent cells: Remove growth medium from the cells and add 1.0ml of RNAifectin
TM -RNA solution to the each well containing cells. Incubate at 37˚C.
5b.Suspension cells: Add 0.2ml of the RNAifectin
TM -RNA solution into each well containing suspension cells in 0.8ml serum-free, antibiotic-free medium. Incubate at 37˚C.
6. After 5-8 hours, remove transfection solution and add 2.0ml of the appropriate growth medium (with serum and antibiotics) or add 0.1ml of FBS directly into each vessel. Incubate the cells at 37˚C for a total of 18-24 hours.
7. Assay cell extracts for marker gene activity 24-72 hours after the start of transfection depending on cell type and promoter activity.
8. A similar procedure can be used to transfect an RNAi vector for stable expression. Seventy-two hours after transfection passage the cells 1:10 or higher into an appropriate selective medium.
Optimizing transfection for specific cell lines To achieve the maximum transfection efficiency and low cytotoxicity, optimize transfection conditions by varying cell density along with RNA oligo and RNAifectin
TM concentrations. Optimal results have been observed when cells are 70-90% confluent and RNA(μg): RNAifectin
TM (μl) ratios are 1:1 to 1:5.
Equipment 