Hello Friends,
In a research they calculated (by a microscope) the efficiency of EGFP-tagged plasmid, but they didn't mention the way of calculating. What is the possible ways to carry out this procedure?
Thank you in advance.
It depends on what do you mean by efficiency.
If you refer to the transfection efficiency than you can count the number of GFP positive cells and calculate the fraction of GFP+/total cell number.
If you mean something to do with expression levels than there are softwares that can calculate that out of microscope images.
You could use IMAGEJ to calculate the fluorescence intensity. There is a very comprehensive tutorial in their website. The software is free. There is a ton of stuff you can do with this software.
Link: https://imagej.nih.gov/ij
Do you mean checking the transfection efficiency? There are many ways even with microscope. We prefer to use flow cytometer or a fluorescence cell counter. By microscopy, you can use area coverage but not very reliable. One may also do Hoechst staining GFP colocalization but you need to have a software to do that. Also, it may be hard when you have cells in clumps or colonies. With cell suspension in flow cytometry and fluorescence cell counter, you will get more accurate counting.
Yoav Lubelsky Ramanathan Saikishore Bill Chi Shun Ho thank you all for these helpful information.
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