Abstract
BackgroundTechnical advances following the Human Genome Project revealed that high-quality and -quantity DNA may be obtained from whole saliva samples. However, usability of previously collected samples and the effects of environmental conditions on the samples during collection have not been assessed in detail. In five studies we document the effects of sample volume, handling and storage conditions, type of collection device, and oral sampling location, on quantity, quality, and genetic assessment of DNA extracted from cells present in saliva.MethodsSaliva samples were collected from ten adults in each study. Saliva volumes from .10-1.0 ml, different saliva collection devices, sampling locations in the mouth, room temperature storage, and multiple freeze-thaw cycles were tested. One representative single nucleotide polymorphism (SNP) in the catechol-0-methyltransferase gene (COMT rs4680) and one representative variable number of tandem repeats (VNTR) in the serotonin transporter gene (5-HTTLPR: serotonin transporter linked polymorphic region) were selected for genetic analyses.ResultsThe smallest tested whole saliva volume of .10 ml yielded, on average, 1.43 ± .77 μg DNA and gave accurate genotype calls in both genetic analyses. The usage of collection devices reduced the amount of DNA extracted from the saliva filtrates compared to the whole saliva sample, as 54-92% of the DNA was retained on the device. An "adhered cell" extraction enabled recovery of this DNA and provided good quality and quantity DNA. The DNA from both the saliva filtrates and the adhered cell recovery provided accurate genotype calls. The effects of storage at room temperature (up to 5 days), repeated freeze-thaw cycles (up to 6 cycles), and oral sampling location on DNA extraction and on genetic analysis from saliva were negligible.ConclusionsWhole saliva samples with volumes of at least .10 ml were sufficient to extract good quality and quantity DNA. Using 10 ng of DNA per genotyping reaction, the obtained samples can be used for more than one hundred candidate gene assays. When saliva is collected with an absorbent device, most of the nucleic acid content remains in the device, therefore it is advisable to collect the device separately for later genetic analyses.
Highlights
Technical advances following the Human Genome Project revealed that high-quality and -quantity DNA may be obtained from whole saliva samples
Genotyping Among the 10 subjects, all 3 genotypes were represented at both polymorphisms: COMT Met/Met (n = 3), Val/ Met (n = 5), Val/Val (n = 2); 5-HTTLPR S/S (n = 1), S/L (n = 6), L/L (n = 3)
When saliva is collected via absorbent devices, the majority of the nucleic acid content remains in the device and is not expressed in the saliva filtrate
Summary
Technical advances following the Human Genome Project revealed that high-quality and -quantity DNA may be obtained from whole saliva samples. In five studies we document the effects of sample volume, handling and storage conditions, type of collection device, and oral sampling location, on quantity, quality, and genetic assessment of DNA extracted from cells present in saliva. The past two decades have witnessed the adoption and integration of minimally invasive measures (in saliva) in studies of the psychobiology of the stress response (e.g., cortisol, alpha-amylase [6]), secretory products of the endocrine systems (e.g., testosterone [7]), inflammatory processes (e.g., cytokines [8]), and pathogen-specific antibodies (e.g., HSV [9]) Access to these tools is widespread, collection and assay protocols are being standardized, and salivary analytes and biomarkers are being employed across many subfields
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