Hello,
I am looking for a method to detect the strength of expressed eGFP signals into cells other than qPCR, I am using the UV microscope to recognize the fluorescence but want to determine the strength also , any suggestions ???
Thanks ..
Hi Eman,
The answer depends on if you want a quantitative or qualitative level of expression. For quantitative, you will need qPCR, Western blot, or a fluorimeter/plate reader against a standard with known concentration.
For qualitative, you can compare to another cell line expressing eGFP at a known amount. In that case you can use any method you like as long as the parameters are constant between the known and unknown samples.
Hope this helps!
I suppose you want absolute quantitive data, because relative quantitative data can obviously be acquired by any device that provides (reasonably linear) output on fluorescence intensity, such as a fluorometer, fluorescence plate reader, or a fluorescence microscope equipped with a digital camera.
It depends a bit if you want expression quantified at a single cell level or just at a cell population level, in other words, if it sufficient for you to know the mean eGFP expression or if you require a distribution of expression levels.
For the population only level expression level proceed as follows:
1) Purchase known amount of eGFP.
2) Prepare samples with a known number of non-transfected cells and eGFP-expressing cells. [E.g. five samples of the non-transfected cells and your transfected sample.]
3) Lyse the cell batches from the phase 2 with a nondenaturing lysis buffer.
4) Add your purchased eGFP to the samples with non-transfected cells in varying amounts to produce a standard curve.
5) Measure fluorescence intensity from the cell lysates (with added eGFP and from your unknown).
6) Use the standard curve to establish the amount of eGFP in your eGFP-expressing cell sample.
For the single cell level expression (i.e. the distribution of expression), the method of choice is obviously flow cytometry with your cells and with microspheres containing known amounts of eGFP (e.g. http://www.ebiotrade.com/emgzf/clo2002apr/FACS-EGFP.pdf). Then you get wonderful statistics easily. If you do not have access to a flow cytometer, then you have two options (A & B) available with a fluorescence microscope. AT ALL POINTS, BE SURE TO AVOID SIGNAL SATURATION.
1A) Nevertheless purchase the eGFP-microbeads, 1B) purchase just eGFP.
2A) Image (preferably) hundreds or microbeads of each intensity set with a fluorescence microscope. Segment microbeads from the background. Use the particle area-integrated(or in confocal volume-integrated) fluorescence intensities to recover the distribution of fluorescence intensities of the microbeads. Create a standard curve. 2B) Do the population only level expression measurement described earlier to recover the mean expression level in your cell population.
3AB) Image hundreds of cells from your eGFP-expressing cell population. Define the cells in images, calculate area-integrated (or for confocal volume-integrated) intensity for each cell. Then 3A) match the intensities to the standard curve from the mean intensities of microbeads, or 3B) match the mean intensity to the mean intensity of the population experiment, and assume linearity of the intensity readings.
Thanks Andrew for your answer. In fact, I am trying to compare different promoters for expression eGFP, which is better. Unfortunately, I don't have an expressing model with a known level. But I will follow your suggestions for the future.
Thanks again!
Thanks Juha-Matti for your detailed answer. Yes, I am using fluorescent microscope at the moment and I need to determine the strength of different promoters. I will try do what you suggested for future work.
Thanks again!
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