Anyone know the problem? why the Ct values for my samples are fluctuating so much and even not reaching threshold line? is this related to my unspecified primer? because after I checked my primer again, It could bind several genes outside of my target gene. Then shouldn't the non-specific amplification show a false positive? or high fluorescence level, can non-specific primers also cause low fluorescence? I'm sure it's not the DNA sample / template that's the problem, because when I used another primer (GAPDH specific), the results of the amplification curve and melt curve were satisfactory.
Look at the Y axis units, they should be logarithmic (in RFU) if you were getting good deal of amplification. You could just be looking at background signal. Compare the Y axis units to the amplifications that have sigmoidal curves and there should be a big difference.
You are getting 0 amplification from these primers. If you included a positive control, the scale on the Y-axis would make it obvious that what you are seeing is just background noise.
Thank you for the answer, is this amplification failure due to my unspecified primary? because I have tried it at several annealing temperatures, but the results are still negative, I thought that non-specific primers would only produce false-positives
Even if you were getting non-specific amplification, you would be getting a decent sigmoidal-looking amplification curve. Look at the fluorescence units, they are way too low and non specific amplifications don't behave like ventricular fibrillation ECG traces.
Is it possible that the amplification failure products can be visualized in electrophoresis? Due to the failed amplification results it shows bands in my electrophoresis with bands that are quite clear.
Are you using SYBR green? Anything that would show up in an electrophoresis should also show up on the real time results.
Yes, i am using SYBR green, is the band that appears on my electrophoresis SYBR green? although the band appears clean at 100 bp and 300 bp
but the band appears only at annealing temperatures of 58 and 62, at other annealing temperatures that I tested the band does not appear
Are the electrophoresis bands your expected amplicon sizes for your region of interest? If not, then they are not your amplicon of interest. Just looking at your real time data I can tell that your primers aren't working. I would focus on that part, getting the primers to work. Even non-specific amplicons would provide a nice S or sigmoidal shape amplification graphs.
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