I'm currently doing an experiment where I'm following the expression of a reporter gene (GFP) along growth using S. cerevisiae. The cultivations last around 48 hours. I observed that the results from run to run are very reproducible, in terms of trend/curve shape. However, I cannot say the same regarding the fluorescence values. For the calculations, I subtract the GFP autofluorescence with the correspondent non-GFP expressing strain, and then normalize according to O.D. The settings of the microplate reader are also the same (same gain function, etc). However, even maintaining all the parameters, I get, in terms of number values, different results from run to run (for instance, for the same strain, in one run the fluorescence values normalized are 20000 for one run, and 12000 for another run, althought the curve has precisely the same shape from run to run). I guess that this means that I need a kind of a standart that I know the fluorescence will be the same from run to run, and always need to normalize the GFP values from cells according to that standart. But what should I use?
Thank you!
I'm assuming the fluorescence values are output as "relative fluorescence units" (RFU) which is a pretty arbitrary unit that can be normalized between runs. This thread has some good suggestions:
https://www.researchgate.net/post/What_is_the_meaning_of_Relative_Fluorescence_Unit_RFU_at_a_particular_range_of_wavelength2
Basically after all your other adjustments (auto-fluorescence subtraction, OD standardization) you can normalize all the measurements to the highest value achieved by that run. It sounds like that could help normalize the values while maintaining the trend.
Also, keep in mind there's usually a decent amount of natural variability between biological replicates anyway. This can be compounded in fluorescent protein reporters because their output is affected by a lot of factors including oxygen concentration in each well and scattering/absorbance of fluoresced light by the yeast cells (which may or may not be linearly correlated with the optical density), etc.
Hello Alexandra, thank you so musch for your answer. When you mean the highest value of the run, it's the highest in the plate or the highest of it's own curve?
Assuming you want to preserve differences in the fluorescence intensity between different samples, the highest value in the plate. Depending on how you want to do it you could either make this the value of a single well or if you have replicates you could take an average of your highest sample at its peak fluorescence.
You could also start including GFP under control of a well characterized strong constitutive promoter and make everything relative to that, but that wouldn't work for previously gathered data obviously. How you do this sort of depends on what question(s) you're asking.
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