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ct value of neg

qPCR ct value neg control

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#1 ImNew



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Posted 03 November 2011 - 03:16 AM

Dear guys,

There isn't any amplification of neg control in the qpcr; meaning there isn't any fluorescence signal for the NTC at all (straight line throughout 40 cycles).

Does it mean that the CT value of the NTC is zero? And by the way, what does ct value means? Is it even possible to get a ct value of zero for NTC?



#2 Archie



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Posted 03 November 2011 - 04:12 AM

The Cq (quantification cycle), Ct (threshold cycle), Cp (crossing point) or TOP (take-off point) is defined as the number of cycles that is required for the fluorescent signal to cross a certain threshold.
This threshold can be set by yourself or the software you're using and should exceed the background signal and lie in the exponential phase of the reaction as well. The values are inversely proportional to the amount of target in your sample what means that the lower the Cp the greater the amount of target nucleic acid.

If your NTC reaction doesn't give you a signal then there seems to be no (un-)specific reaction within the range of cycles you're measuring.
An NTC is either negative or positive with a certain CT but not zero.
If it is positive there could be several reason depending on the chemistry you're using (e.g. SYBR Green, probe-based assays): unspecific amplification, formation of primer-dimers, specific amplification due to contamination etc

Edited by Archie, 03 November 2011 - 04:13 AM.

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