Focus on methodology: salivary bioscience and research on adolescence: an integrated perspective.

The characterization of the salivary proteome and advances in biotechnology create an opportunity for developmental scientists to measure multi-level components of biological systems in oral fluids and identify relationships with developmental processes and behavioral and social forces. The implications for developmental science are profound because from a single oral fluid specimen, information can be obtained about a broad array of biological systems and the genetic polymorphisms related to their function. The purpose of this review is to provide a conceptual and tactical roadmap for investigators interested in integrating these measurement tools into research on adolescent health and development.

Assessing genetic polymorphisms using DNA extracted from cells present in saliva samples.

BACKGROUND: Technical 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. METHODS: Saliva 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. RESULTS: The 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. CONCLUSIONS: Whole 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.

Salivary flow and alpha-amylase: collection technique, duration, and oral fluid type.

There has been renewed interest in salivary alpha-amylase (sAA), a surrogate marker of autonomic/sympathetic activity, in biosocial research on stress vulnerability, reactivity, and recovery. This study explored the impact of saliva flow rate on sAA measurement by examining the influence of (1) the technique used to collect oral fluid-synthetic swab, cotton pledget, hydrocellulose microsponge, or passive drool; (2) collection point duration--the length of time the technique is employed (1-5min); and (3) oral fluid type--whole unstimulated saliva (not absorbed by any material) or oral fluid sampled from areas near the parotid, submandibular, or sublingual salivary glands. sAA activity (U/mL) was the highest in oral fluid collected from the parotid and submandibular gland areas. The volume (mL) of oral fluid collected increased, and the activity of sAA (U/mL) decreased, as collection point duration lengthened. The magnitude of these effects varied according to collection technique and oral fluid type. Across all conditions, there were positive correlations (range .70-.88) between sAA activity (U/mL) and sAA output (U/min). Management of these potential sources of measurement error will be essential to ensuring the success of future research on the correlates and concomitants of sAA activity, stress-related reactivity and recovery, and diurnal variation.