Clinical trial participant characteristics and saliva and DNA metrics.

Author(s): Nishita DM, Jack LM, McElroy M, McClure JB, Richards J, Swan GE, Bergen AW

Publication: BMC Med Res Methodol, 2009, Vol. 9, Page 71

PubMed ID: 19874586 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of this study was to determine the effects of patient demographics, storage, centrifugation speed (2500 x g versus 3000 x g), and tech platform on the assessment of DNA yield and genotyping success in saliva specimens from cigarette smokers. 565 saliva specimens were self-collected using Oragene DNA Self-Collection Kits and mailed back to the laboratory. Only a percentage of the isolated DNA was identified to be human.

   Summary of Findings:

   High correlations in DNA yield measurements were obtained by spectrophotometer and PicoGreen (r=0.922, p<0.001), spectrophotometer and real-time PCR (r=0.832, p<0.001), and by PicoGreen and real-time PCR (r=0.962, p<0.001). However, the average DNA yields were 155.2 ug when assessed by spectrophotometer, 82.8 ug when measured by PicoGreen, and 61.5 ug when measured by real-time PCR. DNA yields were also weakly to moderately correlated with specimen volume (r<0.25 for all three methods), and weakly inversely correlated with the calculated percentage of human DNA (r=-0.148, p=0.005). DNA yield as assessed by each of the three methods was not affected by storage at room temperature, but the DNA purity, as assessed by spectrophotometry, decreased in specimens stored for more than 55 days compared to specimens stored for less than 55 days. Compared to specimens provided by males, females provided smaller specimen volumes with lower DNA yields by spectrophotometer, but the saliva had a higher clarity and the resultant DNA had a higher percentage of human DNA as opposed to DNA from other sources. Increased patient age was associated with reduced saliva clarity and a lower percentage of human DNA. The authors report that centrifugation speed during DNA extraction did not impact DNA yield or genotyping success, and the number of cigarettes smoked per day did not affect DNA yield or the percentage of human DNA. Template DNA amount was not associated with SNP TaqMan genotyping success but was associated with VNTR completion rates (p<0.0001), but the amounts tested were not specified by the authors. The completion rates for the 11 single nucleotide polymorphisms were over 98% for each subject, and the completion rates for variable number tandem repeats were 98.5% and 97.5% for each subject for SLC6A3 and DRD4, respectively.

   Biospecimens

   • Bodily Fluid - Saliva

   Preservative Types

   • None (Fresh)

   Diagnoses:

   • Not specified

   Platform:

   Analyte	Technology Platform
   DNA	Fluorometry
   DNA	Real-time qPCR
   DNA	Microsatellite analysis
   DNA	Spectrophotometry
   DNA	SNP assay
   Morphology	Macroscopic observation

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Preaquisition	Patient age	19-76 years
   Preaquisition	Patient gender	Female Male
   Preaquisition	Other drugs	2-80 cigarettes per day
   Storage	Time at room temperature	2-55 days 55-203 days
   Real-time qPCR Specific	Targeted nucleic acid	Intra-ALu based amplicon
   SNP assay Specific	Targeted nucleic acid	11 unspecified TaqMan assays
   Spectrophotometry Specific	Technology platform	PicoGreen Real-time PCR
   Real-time qPCR Specific	Template/input amount	Various unspecified amounts
   Microsatellite analysis Specific	Targeted nucleic acid	VNTR in SLC6A3 VNTR in DRD4
   Biospecimen Aliquots and Components	Centrifugation	Multiple speeds compared

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