Dear all,
We have tried to transfect MDA-MB-231 by lipofectamine 2000. However, the transfection rate was below 2%.
Recently, we transfected using electroporation (Eppendorf device).
For the first time, the transfection rate was approx 70% after 24h. The electroporation condition was: Voltage: 30 V, cuvette : 2 mm , pulse: 4 , time: 40 μs , vector : pIRES2-EGFP , vector size : 5700 bp
But in the next attempt, no transfection achieved whatsoever. Thus, we have tried various voltage with different pulses values, but still, we did not achieve any result.
Sincerely seek your kind suggestion.
That's interesting. I am working a lot with transfection either by Lipofectamine or Electroporation. I don't have any problems with both protocol. I need more information on your protocol.
Usually the quality of DNA must be ensured as well as the cell. Proper amount of DNA is important for successful transfection. Also try to use appropriate controls or other DNA to compare.
What is the passage of your cell? Did you grow new cells for transfection? How did you prepare your cells before transfection by electroporation?
Thank you so much for kind help.
Does the passage number of the cell cause NO transfection at all?
I don’t know how many passage have the cells passed before I purchased it. But during the electroporation it thinks it was passage 10.
Honestly, in the next attempts, I found some changes in the morphology of the cells. But I was not sure whether these changes could reduce the transfection rate to 0 !
About the DNA, I try to prepare fresh DNA. The primary concentration of DNA are usually 0.5 μg/ml. But the final concentration is 15 μg/ml.
Dear Dr. Edward,
Do you have any experience with MDA-MB-231 cell line? May I know the electroporation condition? The voltage, pulse and time?
I followed the Eppendorf protocol for electroporation as below:
1. Dilute the cells in culture medium with 0.5% FBS and determine the number of cells
2. Resuspend the cells in Electroporation Buffer (at RT) and set a cell concentration of 1 x 106 cells/ml
3. Aliquot the cell suspension (400 μl for a cuvette with 2-mm gap in Eppendorf tubes)
4. Add plasmid DNA (final concentration 15 μg/ml) and mix at RT. (NO incubation time)
5. Transfer the cell suspension to electroporation cuvettes.
5. After pulsing, allow the cell suspension to remain in the cuvette for 10 minutes at RT.
Carefully remove the cell suspension from the cuvette and cultivate it in 3 ml culture medium in a 60-mm culture dish.
Many thanks
MDA-MB-231 is one of those cell lines that is difficult to transfect. We used lipofectamine 2000 (Invitrogen) or the Promega Viafect in the lab, and we only get below or around 50% transfection rate for MDA-MB-231. You can try to use Fugene HD from Promega, it works with cells that are difficult to transfect with high efficiency.
Dear Dr. Rosa,
Thank you so much. Could you please send me your lipofectamine 2000 (invitrogen) protocol, specific for MDA-MB-231 ?
Hey, I am answering your comment point-by-point. Here it is...
Thank you so much for kind help.
Does the passage number of the cell cause NO transfection at all?
I don’t know how many passage have the cells passed before I purchased it. But during the electroporation it thinks it was passage 10.
-->Generally, electroporation gets you a good transfection. As you've experience somewhat better than lipofectamine 2000. And I too had better transfection efficiency using electroporation between 70%-100%
Honestly, in the next attempts, I found some changes in the morphology of the cells. But I was not sure whether these changes could reduce the transfection rate to 0 !
-->When changes in morphology happens, something is wrong with the cell condition already. You really have to be very careful with your protocol. I've experienced it.
About the DNA, I try to prepare fresh DNA. The primary concentration of DNA are usually 0.5 μg/ml. But the final concentration is 15 μg/ml.
-->Are you sure it's µg/ml not µg/µl? Because 0.5 µg/ml is very low. I usually use 0.5-1 µg/µl.
Dear Dr. Edward,
Do you have any experience with MDA-MB-231 cell line? May I know the electroporation condition? The voltage, pulse and time?
-->I'm using different cell lines as well as primary cells but I have never had any trouble transfecting them. The key is really extra caution and care with your cells.
I followed the Eppendorf protocol for electroporation as below:
1. Dilute the cells in culture medium with 0.5% FBS and determine the number of cells
-->I use culture media for counting cells. (Not critical)
2. Resuspend the cells in Electroporation Buffer (at RT) and set a cell concentration of 1 x 106 cells/ml
3. Aliquot the cell suspension (400 μl for a cuvette with 2-mm gap in Eppendorf tubes)
4. Add plasmid DNA (final concentration 15 μg/ml) and mix at RT. (NO incubation time)
5. Transfer the cell suspension to electroporation cuvettes.
5. After pulsing, allow the cell suspension to remain in the cuvette for 10 minutes at RT.
-->I usually directly mix it with the culture medium, pipetting up and down. Did you notice if your cells clumped together? You need to use a syringe.
Carefully remove the cell suspension from the cuvette and cultivate it in 3 ml culture medium in a 60-mm culture dish.
-->The protocol seem reasonable. Always do everything in RT.
-->What are the contents of your culture medium? Did you remove the antibiotic (Penicillin/Strep)? The media should not contain any antibiotic (Unless you have an antibiotic selection marker for mammalian cells). So media with FBS is OK.
-->And also you should not use and re-use electroporation cuvettes more than 5 times. Electric conduction eventually decreases, thus, not able to transfect cells well.
-->I would also suggest that try to use different voltages (start with a very high and low, the gap between these voltages should be wide enough to estimate your optimum voltage (unless there are papers for reference, if not, find a paper using a cell/cells that is very close to your cell lines), then work your way by finding out the optimal voltage) with constant capacitance and resistance (if you have any - usually the Biorad Xcell has).
Many thanks
-->You're welcome
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