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

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

doi:10.1093/nar/gks203

Cited by 368 publications

(345 citation statements)

References 46 publications

(59 reference statements)

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“…Advantages dPCR principally offers advantages when considering the identification of the presence and abundance of rare sequence mutations (19 ) and in the quantification of nucleic acids (20 ), while also enabling the cis/trans relationships (21,22 ) to be determined. Quantitative and qualitative measurements offer different challenges when considering error, and many of the challenges that befall legacy PCR and qPCR when designing and optimizing an analytically sensitive and specific method directly apply to dPCR; i.e., a nonspecific and/or poorly optimized set of primers may bind the wrong target, leading to a false-positive signal.…”

Section: Routine Adoption Of Quantitative Clinical Molecular Measurementmentioning

confidence: 99%

Considerations for Digital PCR as an Accurate Molecular Diagnostic Tool

2015

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BACKGROUND Digital PCR (dPCR) is an increasingly popular manifestation of PCR that offers a number of unique advantages when applied to preclinical research, particularly when used to detect rare mutations and in the precise quantification of nucleic acids. As is common with many new research methods, the application of dPCR to potential clinical scenarios is also being increasingly described. CONTENT This review addresses some of the factors that need to be considered in the application of dPCR. Compared to real-time quantitative PCR (qPCR), dPCR clearly has the potential to offer more sensitive and considerably more reproducible clinical methods that could lend themselves to diagnostic, prognostic, and predictive tests. But for this to be realized the technology will need to be further developed to reduce cost and simplify application. Concomitantly the preclinical research will need be reported with a comprehensive understanding of the associated errors. dPCR benefits from a far more predictable variance than qPCR but is as susceptible to upstream errors associated with factors like sampling and extraction. dPCR can also suffer systematic bias, particularly leading to underestimation, and internal positive controls are likely to be as important for dPCR as they are for qPCR, especially when reporting the absence of a sequence. SUMMARY In this review we highlight some of the considerations that may be needed when applying dPCR and discuss sources of error. The factors discussed here aim to assist in the translation of dPCR to diagnostic, predictive, or prognostic applications.

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Section: Routine Adoption Of Quantitative Clinical Molecular Measurementmentioning

confidence: 99%

Considerations for Digital PCR as an Accurate Molecular Diagnostic Tool

2015

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“…The development of digital PCR will improve the performance of qRT-PCR, without the use of endogenous or exogenous controls to normalize the results. In contrast, digital PCR is a direct method for quantifying nucleic acids, particularly useful for samples with low abundant miRNAs, and showing a high degree of sensitivity and precision compared to qRT-PCR [17,18]. Finally, next generation sequencing (NGS) platforms, such as those from Solexa/Illumina and 454 Life Sciences/Roche, have been developed to find novel miRNAs, generating a complete expression profile, distinguishing sequentially similar miRNAs and identifying point mutations.…”

Section: Biogenesis and Function Of Mirnasmentioning

confidence: 99%

miRNAs as novel biomarkers in the management of prostate cancer

Filella

Foj

2017

Clinical Chemistry and Laboratory Medicine (CCLM)

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microRNAs (miRNAs) are small non-coding RNAs that control gene expression posttranscriptionally and are part of the giant non codifying genoma. Cumulating data suggest that miRNAs are promising potential biomarkers for many diseases, including cancer. Prostate cancer (PCa) detection is currently based in the serum prostate-specific antigen biomarker and digital rectal examination. However, these methods are limited by a low predictive value and the adverse consequences associated with overdiagnosis and overtreatment. New biomarkers that could be used for PCa detection and prognosis are still needed. Recent studies have demonstrated that aberrant expressions of microRNAs are associated with the underlying mechanisms of PCa. This review attempts to extensively summarize the current knowledge of miRNA expression patterns, as well as their targets and involvement in PCa pathogenesis. We focused our review in the value of circulating and urine miRNAs as biomarkers in PCa patients, highlighting the existing discrepancies between different studies, probably associated with the important methodological issues related to their quantitation and normalization. The majority of studies have been performed in serum or plasma, but urine obtained after prostate massage appears as a new way to explore the usefulness of miRNAs. Large screening studies to select a miRNA profile have been completed, but bioinformatics tools appear as a new approach to select miRNAs that are relevant in PCa development.Promising preliminary results were published concerning miR-141, miR-375 and miR-21, but larger and prospective studies using standardized methodology are necessary to define the value of miRNAs in the detection and prognosis of PCa.

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“…The calculation of CIs of measured template concentration ratios of ddPCR is embedded in the Quantasoft SW and follows published algorithms (15 ). For the calculation of dispersion of the preamplified DNA to genomic DNA and cfDNA ratios the SD from the above-mentioned embedded function of each value was used with respective formulas (16 ) to assess the SD of the ratios, for which data were considered correlated.…”

Section: Statisticsmentioning

confidence: 99%

Digital Droplet PCR for Rapid Quantification of Donor DNA in the Circulation of Transplant Recipients as a Potential Universal Biomarker of Graft Injury

Beck¹,

Bierau²,

Balzer³

et al. 2013

Clinical Chemistry

222

217

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BACKGROUND: Cell-free DNA (cfDNA) from grafts in the circulation of transplant recipients is a potential biomarker of rejection. Its usefulness was investigated after heart transplantation during the maintenance phase by use of microarrays and massive parallel sequencing of donor and recipient DNA. Disadvantages of these methods are high costs, long turnaround times, and need for donor DNA. Therefore, we sought to develop a rapid and cost-effective method using digital droplet PCR (ddPCR).

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

Cited by 368 publications

References 46 publications

Considerations for Digital PCR as an Accurate Molecular Diagnostic Tool

Considerations for Digital PCR as an Accurate Molecular Diagnostic Tool

miRNAs as novel biomarkers in the management of prostate cancer

Digital Droplet PCR for Rapid Quantification of Donor DNA in the Circulation of Transplant Recipients as a Potential Universal Biomarker of Graft Injury

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