I'm trying to transfect adherent human rhabdomyosarcoma (RD) cells with PX458 vectors using Lipofectamine 3000, following the manufacturer's protocol. Here's a brief overview of my procedure:
- Tube A: 50 μl serum-free medium + 1.5 μl Lipofectamine 3000
- Tube B: 50 μl serum-free medium + 1 μg DNA + 2 μl P3000 reagent
- I mix the tubes and incubate at room temperature for 20 minutes.
4. I add the complex dropwise to the wells, incubate for 6–8 hours, then replace the medium with complete culture medium.
5. I wait 48–72 hours before checking for GFP-positive cells.
Despite this, my transfection efficiency is consistently very low (~10%).
Should I wait longer (e.g., 24h) before changing the medium post-transfection? Any suggestions to improve efficiency? I don’t find this low rate normal at all!
Any suggestions to improve efficiency would be greatly appreciated. I've attached microscopy images for reference.
Hello Parsa Dr
There are some suggestions which you may want to consider.
A. Cells are typically not transfected when they are 80% confluent, or close to 100% confluent, because at high confluency, cells exhibit contact inhibition, meaning they stop actively dividing. Actively dividing cells are more efficient at taking up foreign DNA during transfection than stationary or quiescent cells. Moreover, high cell density can create a physical barrier, making it more difficult for transfection reagents to reach all the cells and deliver the DNA effectively. For most cell types, the optimal confluency for transfection is typically between 60% and 80%. This range ensures that cells are actively dividing and not at a stage where contact inhibition has significantly reduced their activity.
B. In transfection, avoiding high DNA concentrations is crucial for maximizing efficiency and minimizing toxicity. Cells have a limited capacity to take up DNA. Excessive DNA can saturate the cellular uptake pathways, reducing the overall efficiency of transfection. In some cases, high DNA concentrations can inhibit the expression of the transfected gene, even if the cells are successfully transfected.
I suggest that you determine the optimal DNA amount for your specific experiment. Start with a small amount of DNA and gradually increase it to find the point where transfection efficiency plateaus or decreases. Ensure your DNA is of high purity and free from contaminants.
C. Serum starvation can create the right environment for the transfection reagents to effectively interact with the cell membrane and deliver the genetic material. Serum proteins in the media can compete with transfection reagents for cell surface receptors needed for entry into the cell. Serum starvation helps synchronize cells in the cell cycle, reducing competition for cell surface receptors, and promoting optimal conditions for the transfection reagents to interact with the cell membrane.
D. You can wait longer than 5-6 hours up to 24 hours before changing the transfection media, especially for less sensitive cells. Some cells are more sensitive to the transfection reagent. For these cells, a shorter incubation period (e.g., 5-6 hours) before media change might be necessary to minimize potential negative effects on cell viability. It is generally recommended to optimize the media change timing for your specific cell type and transfection method.
Hope these suggestions help!
Best,
Parsa Dr , would you provide the specific cell line's name for us? So that our technical team will "Treat the symptoms" more suitable.
Any more details you can also provide together.
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