Hi,
I am designing an assay to measure a polymerase making dsRNA, and I am using a fluorescent dye that glows only when bound to dsRNA, as not ssRNA.
Recently, I have started to include a standard curve in my experiments, to convers the instrument RFUs to something actually useful, but I noticed a worrying trend - fluorescence in the standard samples would start at one level, and then steadily decrease for the first 10-15 minutes (which are critical for the assay), before leveling out. I have attached a graph illustrating the problem.
Does anyone have an idea what might cause this, and how I can counteract this effect?
Thanks in advance!
I suspect that you have some sort of photobleaching going on there. Prolonged excitation of fluorophores is known to damage them. Try to keep your experiment short.
If it´s not photobleaching you may have something settle at the bottom of the cuvette - in this case moderate stirring may help (if possible).
I hope this helps.
Best regards.
I agree that the most likely explanation is photobleaching. This can be reduced by using a smaller number of lamp flashes per measurement and/or taking fewer measurements per unit of time. Different fluorescent dyes have different susceptibilities to photobleaching, so also try more than one dye.
Another possible contributing factor could be loss of the dye to surface adsorption onto the surface of the plate or cuvette. This can be reduced by including a low concentration (e.g. 0.01%) of a nonionic detergent, such as Triton X-100 or Tween-20.
Another possibility is temperature, some intercalating dyes display decreased signal at higher temperatures (at least Qubit dsDNA, I guess it could happen also with others), and the temperature inside the reader tends to increase over time. You could try turning the reader on well in advance and keep the plate inside the reader 15 minutes before starting the measurement.
If the cause is photobleaching, then why does it stop after 10-15 minutes?
Why does the effect decrease and disappear when the concentration decreases?
It is obvious that the reason for the 10-15 minute decrease in fluorescence intensity is not photobleaching.
Thank you everyone for the suggestions!
I would like to provide more details and answer your questions.
1. The dye I am using is Syto9. There is a great paper on this type of assay, that I am basing my work on, but they used mostly endpoint measurements, and I am guessing that's why they haven't observed the effect I am asking about here.
https://doi.org/10.1038/s41598-019-41998-1
2. I thought that the temperature equilibration might be the problem, so I ran the experiments carefully controlling for the temperature, as well as at just room temperature, and the effect persists.
3. The effect does not necessary decrease with the decreased comcentration of sdRNA of my standard, the total difference in RFUs between the start an 15 min mark is roughly 14% for all ds RNA concentrations.
4. After the initial 15-20 min decrease in fluorescense, the readings then remain consistent for as long as 5 hours more.
Maybe I missed it earlier, but from your answer to point 3 I gather that the concentrations (1000nM to 125nM) in the graph are those of the dsRNA. In order to separate whether you lose binding sites for the dye, or the dye itself, can you pre-incubate dsRNA samples without dye for 30min or so, then add the dye?
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