I am using the RNAi starter kit from Qiagen to transfect siRNA into HeLa cells. I follow the fastforward protocol but only got one or two in 200000 cells transfected with the Alexa Fluor labeled control siRNA. The transfected cell was round loosely attached to other heathy cells. I tried not to use serum but still the same bad but with more dead cells. I tried HeLa from other department but still doesn't work. I tried Escort V transfection reagent from Sigma with or without serum but could still only see one or two cells with fluorescence (the cell were abnormally shaped) or none at all (Escort V is more toxic to cells. I tried Escort V using the same protocol as Qiagen except for the preparation of transfection cocktail.). I observed fluorescence usually at the time of 12h or 21h post-transfection. I used RPMI1640(Hyclone) with 10%FBS(Hyclone) and antibiotics.
The protocol is as follow:
Protocol: Fast-Forward Transfection of HeLa cells with siRNA using HiPerFect Transfection Reagent
This protocol has provided successful results when tested in experiments with this cell type. It is provided as a starting point for optimization of siRNA transfection in a 6-well plate.
The amounts given are for one well of a 6-well plate. In this protocol, cell seeding and transfection are performed on the same day.
1.Shortly before transfection, seed 2 x 105 cells per well of a 6-well plate in 2300 µl of an appropriate culture medium containing serum and antibiotics.
2.For the short time until transfection, incubate the cells under normal growth conditions (typically 37°C and 5% CO2). Cells may alternatively be seeded after step 3 of this protocol.
3. Dilute 30 ng siRNA in 100 µl culture medium without serum (this will give a final siRNA concentration of 1 nM after adding complexes to cells in step 5).
Add 12 µl of HiPerFect Transfection Reagent to the diluted siRNA and mix by vortexing.
IMPORTANT: The amount of transfection reagent and siRNA required for optimal performance may vary, depending on the cell line and gene target.
4. Incubate the samples for 5-10 min at room temperature (15-25°C) to allow the formation of transfection complexes.
5. Add the complexes drop-wise onto the cells. Gently swirl the plate to ensure uniform distribution of the transfection complexes.
6. Incubate the cells with the transfection complexes under their normal growth conditions and monitor gene silencing after an appropriate time (e.g., 6-72 h after transfection, depending on experimental setup). Change the medium as required.
Note: The optimal incubation time for gene silencing analysis depends on the cell type, the gene targeted, and the method of analysis. This can be determined by performing a time-course experiment. When using fluorescently labeled siRNA,
microscopic analysis should be performed 4-24 h after transfection.
I hope the Technician Nikky from Qiagen doesn't mind that I post the protocol here.
I don't know what is wrong. The next thing I want to try is to use DMEM as medium. Can you guys help a little? It's been months now.