In my assay microtiter screening I am doing fluorescence measurement using Tecan microplate reader, I found that it is recommended to use the optimal gain function for application that produce results with unknown RFU values (this is the case). In one of the plates that have screened 3 times successively the reader gives me 3 different results with different gains and the problem is with different high rate positions. Is it normal?
An approach to gain setting that I like to use is to set the gain based on the sample with the most fluorescence in order to get about 30-50% of the maximal signal before the detector saturates. That way, all the samples will be on scale, and there is a bit of room at the top end in case it is needed.
An approach to gain setting that I like to use is to set the gain based on the sample with the most fluorescence in order to get about 30-50% of the maximal signal before the detector saturates. That way, all the samples will be on scale, and there is a bit of room at the top end in case it is needed.
Dear Dr Adam,
Thank you very much for your answer. So do I need to use first the optimization function to know the sample with the highest fluorescence and then use this gain for all the samples? May you explain more sir.
Thank again.
If your software has a function that allows for optimization of the gain based on the whole set of wells, then by all means use it. This is not the same as optimization based on a single well, which is what I have seen in some plate reader software. It may be obvious from the design of your experiment which well should have the highest fluorescence. If not, you could set a low gain and read the plate to find out which well has the highest fluorescence, then optimize based on that well.
For the case of comparing data from different runs or plates, the measurement has to be done at identical gain settings. So, for an unknown plate or assay type, first use "optimal gain" to find a good gain value, then afterwards use "manual gain" with te gain setting found during optimal gain procedure. This has also the advantage of faster measurement, as gain optimization takes its time, and you probably don't want to have this time wasted for each measurement.
Actually, some Tecan application specialist should have answered your question, but they are too old-fashioned and out-dated to show up and give help on social websites...
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