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Problem/qPCR, 2 peaks en melting curve

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4 replies to this topic

#1 unam81



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Posted 25 July 2012 - 01:15 PM

Dear all, i have this problem: Two melting peaks on real time PCR and no peaks in the negative controls (NTC OK!!!) this is froma cDNA.

What could be the problem????

Is not dimers from primers because the NTC is Ok.

#2 bob1


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Posted 25 July 2012 - 02:24 PM

Two different products from the PCR - run it on a gel to check.

#3 Kamran



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Posted 26 July 2012 - 09:07 PM

It may be due to residual genomic DNA in your RNA miniprep.

Try to digest your RNA with "RNase-free DNase" and then perform qPCR. Hopefully, you would not have that unwanted peak :)

#4 cecininí



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Posted 09 August 2012 - 04:30 AM

Hi all,

I'm new to qPCR. I have the opposite problem to Unam81. I'm optimizing the concentration of primers. In one of my reference genes, the NTC gives a fluorescence with a Ct of
37, but the minus RT controls gives no signal. I attached two plots of the dissociation curves, the first shows many peaks for NTC but minus RT controls show no product. The second figure shows the curves of a pool of my cDNA samples for different concentrations of primer. Is this a primer-dimer? If yes, Why the peaks present in the NTC, not seen in the -RT controls? and what is happening in the samples? What can I do about it? Should I increase the annealing Tº ?

Thanks to anyone who can help me.dissociation NTC and -RT.jpg dissociation samples.jpg

Edited by cecininí, 09 August 2012 - 04:33 AM.

#5 Trof


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Posted 11 August 2012 - 02:40 AM

cecininí: That can be template-dependent dimers, they only form when there is no template, RT- can be inhibiting the reaction a bit due to the presence of RNA or just primers are binding elsewhere and not together.
Basically if you can clearly distinguish a dimer in NTC from your samples Tm, you can use this. But in your case you have multiple peaks in NTC even in the area of the product Tm. First put the NTC and positive product on gel, to see that is not a contamination, but NTC may not be visible on gel at all.
Second, changing the Tm can affect the dimers as well, so you could at least try to limit their formation around Tm 85, where your product is.

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