Hi,
I try to transfect BHK21 cells with some viral RNAs. the agent I use is to transfect is Lipofectamine MessengerMAX from invitrogen. despite clear and bright fluorescence indicating successful transfection, the overall transfected cells are low in percentage (eyeballed ~10%). I would like to ask what's the reason of low transfection efficiency? is that the transfection agent (my top suspect), or the cell confluency, or just the way it is?
my method:
Capped mRNAs from T7 mMessager mMachine IVT; transfected RNA amount = 5X more than manufacturer's recommend; BHK21 cells at 90% confluency in prior to transfection; Lipofectamine MessengerMAX transfection.
(note: the viral RNA is not infectious to this cell line, meaning it works more like a replicative mRNA, won't escape cells to infect others)
If the cost of the consumables is an issue for you, then using a pre-optimized BHK-21 transfection reagent (https://altogen.com/product/bhk-21-transfection-reagent-kidney-cells-ccl-10/) can help you make sure that you don't have to do separate experimental setups just to get optimal efficiency. 80% from the first go should be easy to get with siRNA transfection, and from there you can culture your cells as necessary.
Hello Tommy,
Transfection always requires optimization rounds. Good is that cells are transfected... to increase efficiency you can try different things
- first you should find the best ratio reagent/mRNA. I do not understand why you used 5x more reagent than what is given as recommendation. I would rather try different amounts of transfection complex to add on cells (with a same ratio, the standard given in the protocol), and second try to use more or less reagent.
- your confluency seems too high. Why not trying to transfect cells before?
- what about your read-out? mRNA effects are observable as early as 2h, maybe good to check between 2 to 6h
- I do not know how effcicient is chemical transfection on BHK21 cells. Here you give some feedback for lipid-based reagent, maybe good to try also a polymer-based reagent (if you want test samples of our Viromers, please ask me, they give pretty good results for fibroblasts).
Hope it helps! Bests, Sandra
BHK-21 Standard Transfection Protocol (24-well plate):
1. Plate 10,000 - 15,000 BHK-21 cells per well in 0.5 ml of complete growth medium 12–24 hours prior to transfection
2. Wash with 1xPBS and add 0.5 ml of fresh growth medium
3. Prepare transfection complexes by mixing 40 µl of serum-free medium, 5.5 µl of transfection reagent, and
• 750 ng DNA (or mRNA), or
• 30 nM - 50 nM of siRNA (or microRNA)
*Referred to a final volume including growth medium
4. Incubate transfection complexes at RT for 15 - 30 minutes
5. Optional: Add 2 µl of Complex Condenser. This reagent reduces the size of transfection complex, therefore increasing transfection efficiency; however it may increase cell toxicity
6. Add prepared transfection complexes to 0.5 ml of complete growth medium with BHK-21 cells (from step 2)
7. Incubate cells at 37ºC in a humidified CO2 incubator
8. Assay for phenotype or target gene expression 48 - 72 hours after transfection
Optional: Transfection efficiency can be increased by addition of Transfection Enhancer reagent. Add 2 µl of Transfection Enhancer reagent 12-24 hours after transfection
If the viability of BHK-21 cells being transfected is affected at 16 - 24 hours post-transfection, the level of cytotoxicity can be decreased by changing the growth medium and eliminating redundant exposure of cells to transfectant
Arvind Singh Chandel, thank you for the protocol. I am quite curious if you know what is the ”Transfection Enhancer reagent” mentioned in your protocol?
If the cost of the consumables is an issue for you, then using a pre-optimized BHK-21 transfection reagent (https://altogen.com/product/bhk-21-transfection-reagent-kidney-cells-ccl-10/) can help you make sure that you don't have to do separate experimental setups just to get optimal efficiency. 80% from the first go should be easy to get with siRNA transfection, and from there you can culture your cells as necessary.
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