Realtime PCR-negative control and dissociation curve! - (Mar/27/2008 )

Hi all,
I did perform Realtime PCR with some samples, include negative and positive control. According to amplification curve, I did not observe the increasing of flourescent signal of negative sample. However, in the dissociation curve, still there was a peak at 80 celcius degree of this sample, same temperature with positive control.
It's quite so strange. Could you please help me with this problem?
And one more thing, in the regative and positive control, from the dissociation curve, I observed 2 peaks at 2 different temperature: 60 celcius degree (negative control) and 80 celcius degree (positive control). And my sample is 70 celcius degree. So, I don know what is the criteria to be sure that it is the negative or positive result?
Please help me in these cases.
Thank you so much for your help.
Thanh Thuy

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Hello Thanh,

What I can say is that you should run your samples on agarose gel to check the product size. Your negative control could have primer-dimers, that is why you have a peak in your melt curve.
How many cycles did you run your realtime? More than 35 cycles could produce primer-dimers. Are your positive controls and samples both DNA or RNA?

Chris

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Thank you Chris for your reply. Actually, I did run the agarose gel before performing realtime PCR. It's not the primer dimer because the product size is 400bps. Because that sample was theoretically considered as the negative sample, so I performed realtime PCR to check it again. Consequently, the amplification curve showed that no signal was detected, but still there is the peak in the melt curve.
The original samples and controls are RNA and were converted to cDNA before Realtime PCR.
Thanks so much,
Thuy

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