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Droplet Digital PCR versus qPCR for gene expression analysis with low abundant targets: from variable nonsense to publication quality data

Scientific reports, 2017-05, Vol.7 (1), p.2409-8, Article 2409 [Peer Reviewed Journal]

Copyright Nature Publishing Group May 2017 ;The Author(s) 2017 ;ISSN: 2045-2322 ;EISSN: 2045-2322 ;DOI: 10.1038/s41598-017-02217-x ;PMID: 28546538

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  • Title:
    Droplet Digital PCR versus qPCR for gene expression analysis with low abundant targets: from variable nonsense to publication quality data
  • Author: Taylor, Sean C ; Laperriere, Genevieve ; Germain, Hugo
  • Subjects: Chemical contaminants ; Contaminants ; Gene Dosage ; Gene expression ; Gene Expression Profiling - methods ; Gene Expression Profiling - standards ; Gene Expression Regulation ; Humans ; Polymerase chain reaction ; Primers ; Real-Time Polymerase Chain Reaction - methods ; Real-Time Polymerase Chain Reaction - standards ; Reference Standards ; Statistical analysis
  • Is Part Of: Scientific reports, 2017-05, Vol.7 (1), p.2409-8, Article 2409
  • Description: Quantitative PCR (qPCR) has become the gold standard technique to measure cDNA and gDNA levels but the resulting data can be highly variable, artifactual and non-reproducible without appropriate verification and validation of both samples and primers. The root cause of poor quality data is typically associated with inadequate dilution of residual protein and chemical contaminants that variably inhibit Taq polymerase and primer annealing. The most susceptible, frustrating and often most interesting samples are those containing low abundant targets with small expression differences of 2-fold or lower. Here, Droplet Digital PCR (ddPCR) and qPCR platforms were directly compared for gene expression analysis using low amounts of purified, synthetic DNA in well characterized samples under identical reaction conditions. We conclude that for sample/target combinations with low levels of nucleic acids (Cq ≥ 29) and/or variable amounts of chemical and protein contaminants, ddPCR technology will produce more precise, reproducible and statistically significant results required for publication quality data. A stepwise methodology is also described to choose between these complimentary technologies to obtain the best results for any experiment.
  • Publisher: England: Nature Publishing Group
  • Language: English
  • Identifier: ISSN: 2045-2322
    EISSN: 2045-2322
    DOI: 10.1038/s41598-017-02217-x
    PMID: 28546538
  • Source: MEDLINE
    PubMed Central
    ProQuest Central
    DOAJ Directory of Open Access Journals
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