I am running NASBA reactions to amplify mRNA. This is done in a qPCR machine (Stratagene Mx3005P). I run the reactions for 2 hours, with readings every 30 seconds (total of 240 readings). Later in the run (so far only once they have plateaued) I am seeing sudden drops in fluorescence approximately every 50 reads (25 minutes). After the drop, the readings continue the same pattern as before but with the fluorescence lower, relative to the drop. So I get what looks like steps at the end of the run. This happens in every reaction well simultaneously leading me to believe that this is an equipment artifact. However, when I run a qPCR on the same machine I do not see this.
As this only happens in the plateau phase, it has not affected my results. However, I am concerned that if I use a sample which has low target concentration, then the stepping might happen during this sample's exponential phase.
My collaborator who is using the same model of machine in a different lab does not have this problem.
Has anyone else seen this, or does anyone have any ideas what might be causing it?
As an employee at equipment manufacturer company (we also have developed some fluorescence readers) I can tell this is probably due to in the machine's inability to measure very bright signal. The fluorescence is being read by a sensor/camera with certain light exposure time, usually set in miliseconds. Those cameras/sensor usually have 3 dynamic ranges or Log10(3). This means by default you can read 10,100,1000 relative fluorescence units, this also means that you can trust the results only from 1-1000 RFU, results bigger than 1000 RFU are out of the dynamic range and 2000 RFU 3000 RFU or even 10000000 RFU are "equally" bright for the sensor or if to say simply, fluorescent light is blinding the sensor, so if you see a plateaued this can mean that the machine cannot see more. But manufacturers, in the pursuit of improving specifications of their machines, are playing with the exposure time, so if the sample is too bright they just switch to a lower exposure time, as maybe in your case the sample was so bright that the machine put it on the lower log and did not notice it.
Why it does not happen during qPCR? Cycling is the limiting factor: with each cycle total amount of DNA doubles. But NASBA is an avalanche type reaction and you would receive much more Nucleic Acid in comparison to qPCR, so in a result much brighter fluorescence signal.
But don't worry, the machine is working fine, maybe add less fluorescence marker in your sample.
That's a very interesting answer and something that I would not have thought of. Thank you very much.
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