2 replies to this topic

[shijicai]

Hi, I've carried out a qRT-PCR, and there are two peaks generated by NTC on the melting curve.
Can any body tell me what do the two peaks represent? And how can I confirm this?
I've attached the image below.
Thx

Attached Thumbnails

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[Trof]

First, it would be helpful to descibe what other lines in the image means, there are two sorts of peaks, 84 and 91 C single peaks and your NTC one. It it the NTC from the 91 C product? If so, you have probably a slight contamination in NTC and maybe primer dimers. Is this the first time you ran your samples, or was the NTC always a straight line before?

You can run the NTC and samples on gel to see if there is really contamination with your product and if the peak with lower temperature is a dimer.

[shijicai]

Trof, on 25 May 2011 - 07:36 AM, said:

First, it would be helpful to descibe what other lines in the image means, there are two sorts of peaks, 84 and 91 C single peaks and your NTC one. It it the NTC from the 91 C product? If so, you have probably a slight contamination in NTC and maybe primer dimers. Is this the first time you ran your samples, or was the NTC always a straight line before?

You can run the NTC and samples on gel to see if there is really contamination with your product and if the peak with lower temperature is a dimer.

yes, this is my first time.
There are to kinds of mrna in the qPCR,

91C would be the peaks for Metallothionein(MT) mRNA
84C are the peaks for GAPDH.

we treated them by using three different reagents + an untreated one.
Cad, Dex, PMA


The NTC is the NTC for MT.

Thank you