When we measured RNA concentration using fluorescence method with Modulus Turner Biosystems and measured the same sample a few times in a row without opening the lid, sometimes the fluorescence was decreasing but sometimes increasing and after reaching the highest fluorescence decreasing. Why is that so?
Does it mean that we should measure the sample as many times as it reaches the highest fluorescence (concentration) and work with this number?
Which kit/stain do you use ? How long do you incubate the fluorescence dye ? How concentrated is your sample, maybe use dilution of samples? I guess on the one hand with measuring/exciting the dye often you somehow "bleach" it, that for decreasing fluorescence... On the other hand you maybe do not incubate long enough before measuring... So still some dye binds to RNA during measurement and it increases fluorescence.. How large is the increase and/or decrease ? You anyway cannot expect to have exactly the same fluorescence all the time.. Do you also measure a standard with your sample ? This would rule out systematic error bias cause the standard than has the same decrease/increase "effects"
Hello, thank you for the answer. Well, firstly, we use Quant-iT™ RiboGreen® RNA Assay Kit and between adding the dye and measuring it takes about 5 minutes. Secondly, we do not use standards with samples and we diluted samples 1000 - 10 000x before measuring. The thing is, that we thought that by exciting/measuring the dye the fluorescence will decrease, as you wrote we "bleach it", but not increasing.
But I think the problem is the incubation. How long it should take?
Hmm 5 minutes should be ok.. I used the protocol as well and what i can remember it was also 5 minutes... But how you quantify it if you don't use the standard ?
The standard curve was made in advance and is set in the software of Modulus Single Tube Multimode Reader . Standards were a part of Assay kit.
Standards have to prepared each time for each run due to pipetting errors fluorescence reaction and loss of fluorescence due to storing.. Maybe this explains your problems
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