Here's the situation: I am currently using 6-Well plates & HEK293t cells with DMEM +10% FBS + 1% P/S and OptiMEM w/ Lipofectamine 2000 for my transfection. Before transfection when cells are 60-75% confluent, I usually change media by adding 2 mL fresh, warm media via 1 mL pipet (therefore 2x). But when doing so, cells detach very easily from the edges of the wells. Probably about 75% stay attached, but these are more localized to the center. I am transfecting in a specific plasmid at low concentration, so I am worried that this detachment will cause lower transfection efficiency in my cells and this plasmid won't get expressed due to low input. Should I redo these replicates?
Hi,
I used to face a similar problem before.
Regarding checking whether the problem can affect transfection efficiency, you can perform qPCR to assess the expression of your target gene after transfection. If the expression level of the target is acceptable on the desired scale, the problem might not significantly affect the results.
However, the detachment of the cells will indeed influence the transfection efficiency (refer to the link below). Thus, I'd like to suggest several following ways to minimize the problem:
https://www.gbo.com/fileadmin/media/USA/01_Downloads_BioScience/SALES_Scientific_Publications/F073103_AN_Transfection_Efficiency_CELLCOAT.pdf
I hope this information is useful for you.
Good luck!
Phan Thi Lam Hong Thank you for this information. I'm transfecting in a 2-point mutant of an abundantly expressed gene in mammalian cell lines, so qPCR would not be too effective because it would tell me total signal and is not selective to this specific mutant. I guess i could design primers for this exogenous transcript specifically but I worry about non-specific binding to endogenous material and therefore I couldn't be certain which signal is which... thoughts?
I've seen a lot of good things about coating plates. I might try that next. I handle media changes and transfection medium application very carefully, so I can't improve that. I also transfect at 75% confluency or so, any higher density might be a hindrance to transfection efficiency rather than help it due to tight cell-cell contact and clumping.
Regarding assessing the expression of specific mutants, I typically refer to primers used in several papers that describe the design of specific primers for detecting mutant expression. In addition, you can consider to perform RT-qPCR using two sets of primers: one targeting the wildtype sequence, and the other targeting the mutant sequence.
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