International Comparison of Enumeration-Based Quantification of DNA Copy-Concentration Using Flow Cytometric Counting and Digital Polymerase Chain Reaction
| Autor: | Janaína J. V. Cavalcante, Bruno Cosme, Sang-Ryoul Park, Zhiwei Sui, Muslum Akgoz, Mátrai Janka, Alexandra S. Whale, Kerry R. Emslie, Lianhua Dong, Mojca Milavec, Jing Wang, Jim F. Huggett, Michael Forbes-Smith, Jacob L H McLaughlin, Erkan Mozioğlu, Daniel G. Burke, Helen C. Parkes, Jo Lynne Harenza, Margaret C. Kline, Philippe Corbisier, Roberto Becht Flatshart, Jernej Pavšič, Hee-Bong Yoo, Peter M. Vallone |
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| Rok vydání: | 2016 |
| Předmět: |
0301 basic medicine
Chromatography Nucleotides Chemistry Absolute quantification Analytical chemistry Electrophoresis Capillary DNA Flow Cytometry Polymerase Chain Reaction Mass Spectrometry Analytical Chemistry 03 medical and health sciences chemistry.chemical_compound 030104 developmental biology Capillary electrophoresis Enumeration Calibration Digital polymerase chain reaction Plasmids |
| Zdroj: | Analytical Chemistry. 88:12169-12176 |
| ISSN: | 1520-6882 0003-2700 |
| DOI: | 10.1021/acs.analchem.6b03076 |
| Popis: | Enumeration-based determination of DNA copy-concentration was assessed through an international comparison among national metrology institutes (NMIs) and designated institutes (DIs). Enumeration-based quantification does not require a calibration standard thereby providing a route to "absolute quantification", which offers the potential for reliable value assignments of DNA reference materials, and International System of Units (SI) traceability to copy number 1 through accurate counting. In this study, 2 enumeration-based methods, flow cytometric (FCM) counting and the digital polymerase chain reaction (dPCR), were compared to quantify a solution of the pBR322 plasmid at a concentration of several thousand copies per microliter. In addition, 2 orthogonal chemical-analysis methods based on nucleotide quantification, isotope-dilution mass spectrometry (IDMS) and capillary electrophoresis (CE) were applied to quantify a more concentrated solution of the plasmid. Although 9 dPCR results from 8 laboratories showed some dispersion (relative standard deviation [RSD] = 11.8%), their means were closely aligned with those of the FCM-based counting method and the orthogonal chemical-analysis methods, corrected for gravimetric dilution factors. Using the means of dPCR results, the RSD of all 4 methods was 1.8%, which strongly supported the validity of the recent enumeration approaches. Despite a good overall agreement, the individual dPCR results were not sufficiently covered by the reported measurement uncertainties. These findings suggest that some laboratories may not have considered all factors contributing to the measurement uncertainty of dPCR, and further investigation of this possibility is warranted. |
| Databáze: | OpenAIRE |
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