title: Direct elicitation of template concentration from quantification cycle (Cq) distributions in digital PCR.
abstract: 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.
authors.affiliation: Department of Biological Sciences, University of Calgary, Calgary, AB T2N 1N4, Canada.
authors.forename: Mitra
authors.initials: M
authors.lastname: Mojtahedi
authors.affiliation: Institute for Systems Biology, 401 Terry Ave N, Seattle, WA 98109, USA Luxembourg Centre for Systems Biomedicine, L-4362 Esch-sur-Alzette, Luxembourg aymeric.dherouel@uni.lu.
authors.forename: Aymeric
authors.initials: A
authors.lastname: Fouquier d'Hérouël
authors.affiliation: Department of Biological Sciences, University of Calgary, Calgary, AB T2N 1N4, Canada Institute for Systems Biology, 401 Terry Ave N, Seattle, WA 98109, USA sui.huang@systemsbiology.org.
authors.forename: Sui
authors.initials: S
authors.lastname: Huang
mesh_terms: D000818:Animals; D002460:Cell Line; D041905:Erythroid Cells / Q000378:metabolism; D015870:Gene Expression; D006412:Hematopoietic Stem Cells / Q000378:metabolism; D051379:Mice; D022423:Myeloid Cells / Q000378:metabolism; D010957:Plasmids; D016133:Polymerase Chain Reaction / Q000379:methods*; D013698:Templates, Genetic
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