title: Accuracy in copy number calling by qPCR and PRT: a matter of DNA.
abstract: The possible implication of copy number variation (CNV) in the genetic susceptibility to human disease needs to be assessed using robust methods that can be applied at a population scale. In this report, we analyze the performance of the two major techniques, quantitative PCR (qPCR) and paralog ratio test (PRT), and investigate the influence of input DNA amount and template integrity on the reliability of both methods. Analysis of three genes (PRELID1, SYNPO and DEFB4) in a large sample set showed that both methods are prone to false copy number assignments if sufficient attention is not paid to DNA concentration and quality. Accurate normalization of samples is essential for reproducible qPCR because it avoids the effect of differential amplification efficiencies between target and control assays, whereas PRT is generally more sensitive to template degradation due to the fact that longer amplicons are usually needed to optimize sensitivity and specificity of paralog sequence PCR. The use of normalized, high quality genomic DNA yields comparable results with both methods.
authors.affiliation: Immunogenetics Research Laboratory, Cruces University Hospital, Barakaldo, Basque Country, Spain.
authors.forename: Nora
authors.initials: N
authors.lastname: Fernandez-Jimenez
authors.forename: Ainara
authors.initials: A
authors.lastname: Castellanos-Rubio
authors.forename: Leticia
authors.initials: L
authors.lastname: Plaza-Izurieta
authors.forename: Galder
authors.initials: G
authors.lastname: Gutierrez
authors.forename: Iñaki
authors.initials: I
authors.lastname: Irastorza
authors.forename: Luis
authors.initials: L
authors.lastname: Castaño
authors.forename: Juan Carlos
authors.initials: JC
authors.lastname: Vitoria
authors.forename: Jose Ramon
authors.initials: JR
authors.lastname: Bilbao
mesh_terms: D001681:Biological Assay / Q000379:methods*; D004247:DNA / Q000235:genetics*; D056915:DNA Copy Number Variations / Q000235:genetics*; D006801:Humans; D008840:Microfilament Proteins / Q000235:genetics; D024101:Mitochondrial Proteins / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D012689:Sequence Homology, Nucleic Acid; D023083:beta-Defensins / Q000235:genetics
chemical_list: C106641:DEFB4A protein, human; D008840:Microfilament Proteins; D024101:Mitochondrial Proteins; C102194:PRELID1 protein, human; C503967:SYNPO protein, human; D023083:beta-Defensins; D004247:DNA