title: Enhanced analysis of real-time PCR data by using a variable efficiency model: FPK-PCR.
abstract: Current methodology in real-time Polymerase chain reaction (PCR) analysis performs well provided PCR efficiency remains constant over reactions. Yet, small changes in efficiency can lead to large quantification errors. Particularly in biological samples, the possible presence of inhibitors forms a challenge. We present a new approach to single reaction efficiency calculation, called Full Process Kinetics-PCR (FPK-PCR). It combines a kinetically more realistic model with flexible adaptation to the full range of data. By reconstructing the entire chain of cycle efficiencies, rather than restricting the focus on a 'window of application', one extracts additional information and loses a level of arbitrariness. The maximal efficiency estimates returned by the model are comparable in accuracy and precision to both the golden standard of serial dilution and other single reaction efficiency methods. The cycle-to-cycle changes in efficiency, as described by the FPK-PCR procedure, stay considerably closer to the data than those from other S-shaped models. The assessment of individual cycle efficiencies returns more information than other single efficiency methods. It allows in-depth interpretation of real-time PCR data and reconstruction of the fluorescence data, providing quality control. Finally, by implementing a global efficiency model, reproducibility is improved as the selection of a window of application is avoided.
authors.affiliation: Platform for Molecular Biology and Biotechnology, Scientific Institute of Public Health, J. Wytsmanstreet 14, B-1050 Brussels, Belgium. antoon.lievens@wiv-isp.be
authors.forename: Antoon
authors.initials: A
authors.lastname: Lievens
authors.forename: S
authors.initials: S
authors.lastname: Van Aelst
authors.forename: M
authors.initials: M
authors.lastname: Van den Bulcke
authors.forename: E
authors.initials: E
authors.lastname: Goetghebeur
mesh_terms: D018744:DNA, Plant / Q000032:analysis; D005453:Fluorescence; D007700:Kinetics; D016015:Logistic Models; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D013025:Glycine max / Q000235:genetics
chemical_list: D018744:DNA, Plant
references.citation: Math Biosci Eng. 2010 Apr;7(2):363-84
references.pmid: 20462294
references.citation: Biochim Biophys Acta. 2000 Nov 15;1494(1-2):23-7
references.pmid: 11072065
references.citation: Stat Appl Genet Mol Biol. 2007;6:Article10
references.pmid: 17402916
references.citation: BMC Bioinformatics. 2010 Apr 12;11:186
references.pmid: 20385019
references.citation: BMC Bioinformatics. 2008 Feb 12;9:95
references.pmid: 18267040
references.citation: Math Biosci. 2007 Jan;205(1):108-36
references.pmid: 17027039
references.citation: Biotechnol Bioeng. 2005 Sep 30;91(7):848-60
references.pmid: 15986490
references.citation: Biotechnol Bioeng. 2005 Nov 5;92(3):346-55
references.pmid: 16170827
references.citation: J Comput Biol. 2005 Oct;12(8):1047-64
references.pmid: 16241897
references.citation: BMC Mol Biol. 2008 Oct 30;9:96
references.pmid: 18973660
references.citation: Anal Biochem. 2006 Sep 15;356(2):182-93
references.pmid: 16899212
references.citation: J Mol Diagn. 2001 May;3(2):55-61
references.pmid: 11333300
references.citation: Anal Biochem. 2006 Apr 15;351(2):308-10
references.pmid: 16524557
references.citation: Philos Trans R Soc Lond B Biol Sci. 2015 Apr 19;370(1666):
references.pmid: 25750242
references.citation: J Forensic Sci. 2006 Jul;51(4):795-804
references.pmid: 16882221
references.citation: Appl Environ Microbiol. 1997 Oct;63(10):3741-51
references.pmid: 9327537
references.citation: J Theor Biol. 2006 Oct 21;242(4):947-53
references.pmid: 16843498
references.citation: Anal Biochem. 2008 Sep 15;380(2):315-22
references.pmid: 18570886
references.citation: J Theor Biol. 1997 Oct 7;188(3):313-8
references.pmid: 9344735
references.citation: J Theor Biol. 1997 Feb 21;184(4):433-40
references.pmid: 9082073
references.citation: J Theor Biol. 2003 Oct 7;224(3):299-304
references.pmid: 12941588
references.citation: J AOAC Int. 2002 Jul-Aug;85(4):938-44
references.pmid: 12180691
references.citation: Anal Bioanal Chem. 2010 Mar;396(6):1977-90
references.pmid: 19789856
references.citation: BMC Mol Biol. 2007 Dec 20;8:113
references.pmid: 18093344
references.citation: Nucleic Acids Res. 2003 Sep 1;31(17):e105
references.pmid: 12930979
references.citation: Nucleic Acids Res. 2003 Jul 15;31(14):e73
references.pmid: 12853650
references.citation: Mol Aspects Med. 2006 Apr-Jun;27(2-3):95-125
references.pmid: 16460794
references.citation: J Forensic Sci. 2010 Jan;55(1):25-33
references.pmid: 20015162
references.citation: BMC Biotechnol. 2008 May 08;8:47
references.pmid: 18466619
references.citation: Nucleic Acids Res. 2003 Oct 15;31(20):e122
references.pmid: 14530455
references.citation: Nucleic Acids Res. 2009 Apr;37(6):e45
references.pmid: 19237396
references.citation: Anal Biochem. 2002 Mar 1;302(1):52-9
references.pmid: 11846375
references.citation: Forensic Sci Int. 2006 Apr 20;158(1):14-26
references.pmid: 15936161
references.citation: Neurosci Lett. 2003 Mar 13;339(1):62-6
references.pmid: 12618301
references.citation: BMC Bioinformatics. 2007 Mar 09;8:85
references.pmid: 17349040