Please use this identifier to cite or link to this item: https://doi.org/10.1371/journal.pone.0170767
Title: Differences in AMY1 gene copy numbers derived from blood, buccal cells and saliva using quantitative and droplet digital PCR methods: Flagging the pitfall
Authors: Ooi D.S.Q. 
Tan V.M.H.
Ong S.G.
Chan Y.H. 
Heng C.K. 
Lee Y.S. 
Keywords: adult
AMY1 gene
Article
blood sampling
body mass
cheek cell
controlled study
droplet digital polymerase chain reaction
gene
gene dosage
human
informed consent
limit of quantitation
male
polymerase chain reaction
questionnaire
real time polymerase chain reaction
saliva analysis
smoking
adolescent
chemistry
copy number variation
gene expression
genetics
isolation and purification
metabolism
middle aged
mononuclear cell
mouth mucosa
observer variation
polymerase chain reaction
procedures
reproducibility
saliva
standards
alpha amylase saliva isoenzyme
AMY1A protein, human
DNA
Adolescent
Adult
DNA
DNA Copy Number Variations
Gene Dosage
Gene Expression
Humans
Leukocytes, Mononuclear
Male
Middle Aged
Mouth Mucosa
Observer Variation
Polymerase Chain Reaction
Reproducibility of Results
Saliva
Salivary alpha-Amylases
Issue Date: 2017
Publisher: Public Library of Science
Citation: Ooi D.S.Q., Tan V.M.H., Ong S.G., Chan Y.H., Heng C.K., Lee Y.S. (2017). Differences in AMY1 gene copy numbers derived from blood, buccal cells and saliva using quantitative and droplet digital PCR methods: Flagging the pitfall. PLoS ONE 12 (1) : e0170767. ScholarBank@NUS Repository. https://doi.org/10.1371/journal.pone.0170767
Abstract: Introduction: The human salivary (AMY1) gene, encoding salivary ?-amylase, has variable copy number variants (CNVs) in the human genome. We aimed to determine if real-time quantitative polymerase chain reaction (qPCR) and the more recently available Droplet Digital PCR (ddPCR) can provide a precise quantification of the AMY1 gene copy number in blood, buccal cells and saliva samples derived from the same individual. Methods: Seven participants were recruited and DNA was extracted from the blood, buccal cells and saliva samples provided by each participant. Taqman assay real-time qPCR and ddPCR were conducted to quantify AMY1 gene copy numbers. Statistical analysis was carried out to determine the difference in AMY1 gene copy number between the different biological specimens and different assay methods. Results: We found significant within-individual difference (p<0.01) in AMY1 gene copy number between different biological samples as determined by qPCR. However, there was no significant within-individual difference in AMY1 gene copy number between different biological samples as determined by ddPCR. We also found that AMY1 gene copy number of blood samples were comparable between qPCR and ddPCR, while there is a significant difference (p<0.01) between AMY1 gene copy numbers measured by qPCR and ddPCR for both buccal swab and saliva samples. Conclusions: Despite buccal cells and saliva samples being possible sources of DNA, it is pertinent that ddPCR or a single biological sample, preferably blood sample, be used for determining highly polymorphic gene copy numbers like AMY1, due to the large within-individual variability between different biological samples if real time qPCR is employed. © 2017 Ooi et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Source Title: PLoS ONE
URI: https://scholarbank.nus.edu.sg/handle/10635/166025
ISSN: 19326203
DOI: 10.1371/journal.pone.0170767
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