Figure 2: Total DNA yield measured by PicoGreen® vs human DNA yield measured by qPCR, and vs total DNA yield measured by UV, N = 539 COMPASS saliva DNA samples.

Mentions: The overall saliva and saliva DNA characteristics can be seen in Table 1. DNA analysis (N = 539) was limited to one sample per participant for which all three quantification measurements were available. Total DNA yield measurements were all highly significantly correlated (Spearman) (UV vs PG (r = 0.922, P < 0.001), UV vs qPCR (r = 0.832, P < 0.001), and PG vs qPCR (r = 0.962, P < 0.001)) (Figure 2) and saliva volume was significantly correlated with the total DNA yield as measured by each of the three methods using UV, PG, and qPCR (r = 0.33, 0.28, 0.25 respectively, all P < 0.001), but inversely correlated with the calculated % human DNA (r = -0.148, P = 0.005). A median of 77%, a mean (SD) of 76.1% (17.1%), and a range of 2.4-159% estimated human DNA content was obtained (Figure 3). The single sample at 159% was excluded from the figure, but not from analysis. For the sample concentrations for all three quantification methods see Additional file 1.

Nishita DM, Jack LM, McElroy M, McClure JB, Richards J, Swan GE, Bergen AW - BMC Med Res Methodol (2009)

Bottom Line: Significant gender differences were observed with males providing larger saliva volume (4.7 vs. 4.5 ml, p = 0.019), samples that were more likely to be judged as cloudy (39.5% vs. 24.9%, p < 0.001), and samples with greater DNA yield as measured by UV (190.0 vs. 138.5, p = 0.002), but reduced % human DNA content (73.2 vs. 77.6 p = 0.005) than females.Other participant characteristics (age, self-identified ethnicity, baseline cigarettes per day) were associated with saliva clarity.Saliva volume and saliva and DNA clarity were positively correlated with total DNA yield by all three quantification measurements (all r > 0.21, P < 0.001), but negatively correlated with % human DNA content (saliva volume r = -0.148 and all P < 0.010).

Affiliation: Center for Health Sciences, SRI International, Menlo Park, CA, 94025, USA. denise.nishita@sri.com

Abstract: Clinical trial and epidemiological studies need high quality biospecimens from a representative sample of participants to investigate genetic influences on treatment response and disease. Obtaining blood biospecimens presents logistical and financial challenges. As a result, saliva biospecimen collection is becoming more frequent because of the ease of collection and lower cost. This article describes an assessment of saliva biospecimen samples collected through the mail, trial participant demographic and behavioral characteristics, and their association with saliva and DNA quantity and quality.Saliva biospecimens were collected using the Oragene(R) DNA Self-Collection Kits from participants in a National Cancer Institute funded smoking cessation trial. Saliva biospecimens from 565 individuals were visually inspected for clarity prior to and after DNA extraction. DNA samples were then quantified by UV absorbance, PicoGreen(R), and qPCR. Genotyping was performed on 11 SNPs using TaqMan(R) SNP assays and two VNTR assays. Univariate, correlation, and analysis of variance analyses were conducted to observe the relationship between saliva sample and participant characteristics.The biospecimen kit return rate was 58.5% among those invited to participate (n = 967) and 47.1% among all possible COMPASS participants (n = 1202). Significant gender differences were observed with males providing larger saliva volume (4.7 vs. 4.5 ml, p = 0.019), samples that were more likely to be judged as cloudy (39.5% vs. 24.9%, p < 0.001), and samples with greater DNA yield as measured by UV (190.0 vs. 138.5, p = 0.002), but reduced % human DNA content (73.2 vs. 77.6 p = 0.005) than females. Other participant characteristics (age, self-identified ethnicity, baseline cigarettes per day) were associated with saliva clarity. Saliva volume and saliva and DNA clarity were positively correlated with total DNA yield by all three quantification measurements (all r > 0.21, P < 0.001), but negatively correlated with % human DNA content (saliva volume r = -0.148 and all P < 0.010). Genotyping completion rate was not influenced by saliva or DNA clarity.Findings from this study show that demographic and behavioral characteristics of smoking cessation trial participants have significant associations with saliva and DNA metrics, but not with the performance of TaqMan(R) SNP or VNTR genotyping assays.COMPASS; registered as NCT00301145 at clinicaltrials.gov.