Majumdar, Nivedita
(ThermoFisher Scientific, Life Sciences Group, South San Francisco, 94080 USA)
,
Banerjee, Swapnonil
(ThermoFisher Scientific, Life Sciences Group, South San Francisco, 94080 USA)
,
Pallas, Michael
(ThermoFisher Scientific, Life Sciences Group, South San Francisco, 94080 USA)
,
Wessel, Thomas
(ThermoFisher Scientific, Life Sciences Group, South San Francisco, 94080 USA)
,
Hegerich, Patricia
(ThermoFisher Scientific, Life Sciences Group, South San Francisco, 94080 USA)
Digital PCR, a state-of-the-art nucleic acid quantification technique, works by spreading the target material across a large number of partitions. The average number of molecules per partition is estimated using Poisson statistics, and then converted into concentration by dividing by partition volum...
Digital PCR, a state-of-the-art nucleic acid quantification technique, works by spreading the target material across a large number of partitions. The average number of molecules per partition is estimated using Poisson statistics, and then converted into concentration by dividing by partition volume. In this standard approach, identical partition sizing is assumed. Violations of this assumption result in underestimation of target quantity, when using Poisson modeling, especially at higher concentrations. The Poisson-Plus Model accommodates for this underestimation, if statistics of the volume variation are well characterized. The volume variation was measured on the chip array based QuantStudio 3D Digital PCR System using the ROX fluorescence level as a proxy for effective load volume per through-hole. Monte Carlo simulations demonstrate the efficacy of the proposed correction. Empirical measurement of model parameters characterizing the effective load volume on QuantStudio 3D Digital PCR chips is presented. The model was used to analyze digital PCR experiments and showed improved accuracy in quantification. At the higher concentrations, the modeling must take effective fill volume variation into account to produce accurate estimates. The extent of the difference from the standard to the new modeling is positively correlated to the extent of fill volume variation in the effective load of your reactions.
Digital PCR, a state-of-the-art nucleic acid quantification technique, works by spreading the target material across a large number of partitions. The average number of molecules per partition is estimated using Poisson statistics, and then converted into concentration by dividing by partition volume. In this standard approach, identical partition sizing is assumed. Violations of this assumption result in underestimation of target quantity, when using Poisson modeling, especially at higher concentrations. The Poisson-Plus Model accommodates for this underestimation, if statistics of the volume variation are well characterized. The volume variation was measured on the chip array based QuantStudio 3D Digital PCR System using the ROX fluorescence level as a proxy for effective load volume per through-hole. Monte Carlo simulations demonstrate the efficacy of the proposed correction. Empirical measurement of model parameters characterizing the effective load volume on QuantStudio 3D Digital PCR chips is presented. The model was used to analyze digital PCR experiments and showed improved accuracy in quantification. At the higher concentrations, the modeling must take effective fill volume variation into account to produce accurate estimates. The extent of the difference from the standard to the new modeling is positively correlated to the extent of fill volume variation in the effective load of your reactions.
참고문헌 (8)
1. Majumdar N Wessel T Marks J Digital PCR Modeling for Maximal Sensitivity, Dynamic Range and Measurement Precision PLOS One 2015 10 3 1 17 10.1371/journal.pone.0118833
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4. Product pages for QuantStudio 3D Digital PCR system, https://www.thermofisher.com/us/en/home/life-science/pcr/digital-pcr/quantstudio-3d-digital-pcr-system.html (2013).
5. Majumdar N. & Banerjee S. Methods and Systems for Volume Variation Modeling in Digital PCR. US 20160312274 A1 (2015).
6. Huggett J Cowen S Foy C Considerations for Digital PCR as an Accurate Molecular Diagnostic Tool Clinical Chemistry 2015 61 1 79 88 10.1373/clinchem.2014.221366 25338683
7. Corbisier P DNA copy number concentration measured by digital and droplet digital quantitative PCR using certified reference materials Anal Bioanal Chem 2015 407 7 1831 40 10.1007/s00216-015-8458-z 25600685
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