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QPCR replicates issue

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#1 Angeline



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Posted 17 July 2015 - 05:29 AM

Hi. I am new in real time pcr and I have a few doubts.
I would like to do relative comparison on my samples of 3 biological replicates. Is it sufficient to consider only biological replicate in my pcr reaction? Or should I also include In technical replicates? is it advisable to exclude technical replicates and consider only the biological replicate? Thanks.

#2 Mad Researcher

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Posted 21 July 2015 - 02:50 AM

Technical replicate is important and its advisable to do it in triplicates.

Consider the following example:


You get 3 Ct values out of your replicates: 27,3; 27,9; 35. This was pipetted from the same cDNA. So obviously, something wrong happened to the 35 Ct value. You can probably confirm it with the melting curve. And in any case, it is fair (not perfect!) to eliminate your 35 Ct value from your average.


Now imagine you did a 2-plicate. Your values are 27 and 35. What is the good one? You average them? No, the only option would be to set this sample aside from analysis. You just lost a biological replicate to save on a technical replicate.


It is in general far more expensive to generate a biological replicate than a technical one. Do not cut on technical replicates (that is my advice as it is not cheaper anyway). Or at least if you do, expect to have some samples that will be unusable.



Mad Researcher

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