Accuracy in copy number calling by qPCR and PRT: a matter of DNA

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 rati...

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Bibliographic Details
Published in:PloS one 2011-12, Vol.6 (12), p.e28910-e28910
Main Authors: Fernandez-Jimenez, Nora, Castellanos-Rubio, Ainara, Plaza-Izurieta, Leticia, Gutierrez, Galder, Irastorza, Iñaki, Castaño, Luis, Vitoria, Juan Carlos, Bilbao, Jose Ramon
Format: Article
Language:English
Subjects:
Acquired immune deficiency syndrome
AIDS
Analysis
Apoptosis
Basque people
beta-Defensins - genetics
Bioinformatics
Biological Assay - methods
Biology
Celiac disease
Copy number
Deoxyribonucleic acid
Disease
Disease susceptibility
DNA
DNA - genetics
DNA Copy Number Variations - genetics
Gene expression
Genes
Genetic aspects
Genetic diversity
Genetic testing
Genetics
Genomes
HIV
Human immunodeficiency virus
Humans
Laboratories
Medical research
Methods
Microfilament Proteins - genetics
Mitochondrial Proteins - genetics
Nucleotide sequence
Pediatrics
Physical anthropology
Physiology
Polymerase chain reaction
Polymerase Chain Reaction - methods
Reliability analysis
Reproducibility of Results
Sequence Homology, Nucleic Acid
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Summary: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.
ISSN:1932-6203
1932-6203
DOI:10.1371/journal.pone.0028910