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

Delicia Shu Qin Ooi, Verena Ming Hui Tan, Siong Gim Ong, Yiong Huak Chan, Chew Kiat Heng, Yung Seng Lee, Delicia Shu Qin Ooi, Verena Ming Hui Tan, Siong Gim Ong, Yiong Huak Chan, Chew Kiat Heng, Yung Seng Lee

Abstract

Introduction: 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.

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.

Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1. Determination of AMY1 gene copy…
Fig 1. Determination of AMY1 gene copy number by qPCR.
1–7 represents each of the 7 subjects recruited for this study, 8 represents the positive control known to have 14 diploid copies of AMY1 gene. The bars represent the different DNA samples extracted from blood, buccal swab and saliva of each individual. * denotes significant difference of p<0.01 (after bonferroni adjustment) between the different biological samples in each subject using ANOVA analysis.
Fig 2. Determination of AMY1 gene copy…
Fig 2. Determination of AMY1 gene copy number by ddPCR.
1–7 represents each of the 7 subjects recruited for this study, 8 represents the positive control known to have 14 diploid copies of AMY1 gene. The bars represent the different DNA samples extracted from blood, buccal swab and saliva of each individual.
Fig 3. Comparison of AMY1 gene copy…
Fig 3. Comparison of AMY1 gene copy number determined by qPCR and ddPCR in different biological samples.
1–7 represents each of the 7 subjects recruited for this study, 8 represents the positive control known to have 14 diploid copy number of AMY1 gene. The bars represent the 2 different methods used to determine AMY1 gene copy number in the samples (qPCR and ddPCR). A: Comparison of AMY1 gene copy number in blood. B: Comparison of AMY1 gene copy number in buccal swab. C: Comparison of AMY1 gene copy number in saliva. * denotes significant difference of p<0.01 between qPCR and ddPCR for each type of biological sample after paired Student’s t test analysis.

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