Comparison of microfluidic digital PCR and conventional quantitative PCR for measuring copy number variation

Alexandra S Whale, Jim F Huggett, Simon Cowen, Valerie Speirs, Jacqui Shaw, Stephen Ellison, Carole A Foy, Daniel J Scott

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243 Citations (Scopus)
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Abstract

One of the benefits of Digital PCR (dPCR) is the potential for unparalleled precision enabling smaller fold change measurements. An example of an assessment that could benefit from such improved precision is the measurement of tumour-associated copy number variation (CNV) in the cell free DNA (cfDNA) fraction of patient blood plasma. To investigate the potential precision of dPCR and compare it with the established technique of quantitative PCR (qPCR), we used breast cancer cell lines to investigate HER2 gene amplification and modelled a range of different CNVs. We showed that, with equal experimental replication, dPCR could measure a smaller CNV than qPCR. As dPCR precision is directly dependent upon both the number of replicate measurements and the template concentration, we also developed a method to assist the design of dPCR experiments for measuring CNV. Using an existing model (based on Poisson and binomial distributions) to derive an expression for the variance inherent in dPCR, we produced a power calculation to define the experimental size required to reliably detect a given fold change at a given template concentration. This work will facilitate any future translation of dPCR to key diagnostic applications, such as cancer diagnostics and analysis of cfDNA.

Original languageEnglish
Article numbere82
Number of pages9
JournalNucleic Acids Research
Volume40
Issue number11
DOIs
Publication statusPublished - Jun 2012

Keywords

  • Cell Line, Tumor
  • DNA Copy Number Variations
  • DNA, Neoplasm
  • Female
  • Gene Amplification
  • Gene Dosage
  • Genes, erbB-2
  • Humans
  • Microfluidic Analytical Techniques
  • Polymerase Chain Reaction
  • Comparative Study
  • Evaluation Studies
  • Journal Article
  • Research Support, Non-U.S. Gov't

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  • Cite this

    Whale, A. S., Huggett, J. F., Cowen, S., Speirs, V., Shaw, J., Ellison, S., Foy, C. A., & Scott, D. J. (2012). Comparison of microfluidic digital PCR and conventional quantitative PCR for measuring copy number variation. Nucleic Acids Research, 40(11), [e82]. https://doi.org/10.1093/nar/gks203