Reference : Direct elicitation of template concentration from quantification cycle (Cq) distribut...
Scientific journals : Article
Life sciences : Biochemistry, biophysics & molecular biology
Direct elicitation of template concentration from quantification cycle (Cq) distributions in digital PCR
Mojtahedi, Mitra* mailto [University of Calgary - U of C.]
Fouquier d'Hérouël, Aymeric* mailto [University of Luxembourg > Luxembourg Centre for Systems Biomedicine (LCSB) > > ; Institute for Systems Biology - ISB]
Huang, Sui mailto [Institute for Systems Biology - ISB ; University of Calgary - U of C]
* These authors have contributed equally to this work.
Nucleic Acids Research
Oxford University Press
Yes (verified by ORBilu)
United Kingdom
[en] Digital PCR (dPCR) exploits limiting dilution of a template into an array of PCR reactions. From this array the number of reactions that contain at least one (as opposed to zero) initial template is determined, allowing inferring the original template concentration. Here we present a novel protocol to efficiently infer the concentration of a sample and its optimal dilution for dPCR from few targeted qPCR assays. By taking advantage of the real-time amplification feature of qPCR as opposed to relying on endpoint PCR assessment as in standard dPCR prior knowledge of template concentration is not necessary. This eliminates the need for serial dilutions in a separate titration and reduces the number of necessary reactions. We describe the theory underlying our approach and discuss experimental moments that contribute to uncertainty. We present data from a controlled experiment where the initial template concentration is known as proof of principle and apply our method on directly monitoring transcript level change during cell differentiation as well as gauging amplicon numbers in cDNA samples after pre-amplification.

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