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cDNA serial dilution trouble in qPCR: help needed

qPCR primer efficiency

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#1 milko



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Posted 11 November 2015 - 04:11 AM

Hello everybody :)

I need help in understanding some amplification data from qPCR assays.



when I perform amplification (cybergreen-based qPCR) on serial dilutions of cDNA (1:1, 1:10, 1:100) with some (gene) specific primer pairs, I systematically obtain the reversal of starting of the exponential phases: 1:1 related curve spreads AFTER 1:10, or 1:10 AFTER 1:100.

Given that there are no errors in pipetting or diluting... can someone help me in understanding this behaviour?

At the moment, I can only guess that some impurity in the cDNA samples can have random influence on amplification.

Also, I specify that peaks in melting curves looks good and possible shifts are small and not related to primer dimers.

I hope that someone can give me the right hints...



#2 Trof


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Posted 20 November 2015 - 02:58 PM

Less diluted cDNA gives higher Ct? That usually means inhibition.

cDNA may inhibit reaction simply by having too much of (unpurified, since commonly this is not done) RT reaction in the PCR. Recommended maximum volume is 10% of RT in the PCR.

But if it's already diluted 1:1 this seems hardly the case. And the inhibition would be reproducible.

Also, if you do a serial dilution of inhibited sample, you see decrease in Ct with more dilutions, UNTIL the point, where the inhibitor has not further effect, and Cts should be higher with each subsequent dilution as predicted.

So if you have 1:1 Ct higher (later) then 1:10, then 1:100 should be even better or behaving as should, but if you have 1:10 Ct higher than 1:100, 1:1 must be equally inhibited, or even more. 

If the Ct are too high, then it's hardly to tell, there can be small numbers problem too (though that would be just plain random, with lower than expected Ct values in lower concentrations).

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#3 milko



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Posted 23 November 2015 - 03:36 AM

Dear "Brain on a stick", I really thank you very much; your comment made me think productively about the situation :)

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