I performed real time PCR for miRNA. I used SYBR Green Master Mix for miRNA and 5S primers for control. For my samples I should get a peak at 20 cycles. There is no problem in this step. However, in my negative control (water) peak appeared at 41 cycles. I couldn't solve the problem. Would you help me to find what the problem is?
In my experience, any amplification that occur after 38-40th cycles in realtime PCR setting is not reliable and the results are usually not consistent. You can check the melt curve of the PCR product. If the melting temperature of the product in negative control is different from that in your samples, it is a non-specific amplification from any source of contamination.
Hi Emre
It may be some contamination which will build up in some of your reagents over the time...if you are not carefull.....
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