Hi all,
I would like to seek for your opinions on whether fluorescence approach (GFP) or luciferase approach is better in assessing transfection efficiency / success (after miRNA introduction)?
Thank you.
Regards,
Chong
Hello, Just count cells expressing proteins of interest. This is the gold standard method for determining transfection efficiency. Just take one field of view, determine how many cells in total you will count and then see how many are transfected/co-transfected. Do it 3 times to get an average and calc efficiency.
If your plasmids are expressing GFP or another fluorescent protein, you could just put the whole plate under the microscope to count without having to manipulate your cells. Easier and faster.
Alternatively, another fancy method if you have the time and equipment, is to use flow cytometry to sort/count your transfected cells.
Good luck!
Hi,
Fluorescence has always been my go-to for gauging transfection efficiency. A concern with luciferase is that you need to apply your luciferin substrate, wait a few minutes, analyse via plate-reader (etc) which makes it quite a bit more time-consuming.
With GFP (or similar) it's just a case of looking under an fluorescent microscope and comparing % transfected between replicates.
BW
Connor
Hello, Just count cells expressing proteins of interest. This is the gold standard method for determining transfection efficiency. Just take one field of view, determine how many cells in total you will count and then see how many are transfected/co-transfected. Do it 3 times to get an average and calc efficiency.
If your plasmids are expressing GFP or another fluorescent protein, you could just put the whole plate under the microscope to count without having to manipulate your cells. Easier and faster.
Alternatively, another fancy method if you have the time and equipment, is to use flow cytometry to sort/count your transfected cells.
Good luck!
Analyses using a quantitative fluorescence approach, e.g. via FACS analysis (not qualitative, maybe via microscopy) should be favoured in this context, because it will give you quantitative information how many cells were transfected and how strong the target can be blocked - not only how much of an artificial targes signal (vector construct and luciferase expression) is reduced.
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