Differences in AMY1 Gene Copy Numbers Derived from Blood, Buccal Cells and Saliva Using Quantitative and Droplet Digital PCR Methods: Flagging the Pitfall

• Published in: PloS one Vol. 12; no. 1; p. e0170767
• Main Authors: Ooi, Delicia Shu Qin, Tan, Verena Ming Hui, Ong, Siong Gim, Chan, Yiong Huak, Heng, Chew Kiat, Lee, Yung Seng
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 26.01.2017; Public Library of Science (PLoS)
• Subjects: Adolescent; Adult; AMY1 gene; Amylases; Analysis; Analytical chemistry; Biological effects; Biological properties; Biological samples; Biology and Life Sciences; Biomarkers; Blood; Cancer; Children & youth; Copy number; Copy number variations; Deoxyribonucleic acid; Diabetes; DNA; DNA - genetics; DNA - isolation & purification; DNA Copy Number Variations; DNA methylation; Endocrinology; Gene Dosage; Gene Expression; Genes; Genetic engineering; Genetic testing; Genomes; Hospitals; Human genome; Humans; Leukocytes, Mononuclear - chemistry; Leukocytes, Mononuclear - metabolism; Male; Medicine; Medicine and Health Sciences; Methods; Middle Aged; Mouth Mucosa - chemistry; Mouth Mucosa - metabolism; Obesity; Observer Variation; Physiological aspects; Polymerase chain reaction; Polymerase Chain Reaction - methods; Polymerase Chain Reaction - standards; Quality; Real time; Reproducibility of Results; Research and analysis methods; Saliva; Saliva - chemistry; Saliva - metabolism; Salivary alpha-Amylases - genetics; Statistical analysis; Statistical methods; Studies; α-Amylase
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0170767

The human salivary (AMY1) gene, encoding salivary α-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. 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. 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. 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.