A universal pre-analytic solution for concurrent stabilization of salivary proteins, RNA and DNA at ambient temperature.
OBJECTIVE
Saliva is a biofluid that can be obtained from individuals without supervision by health care providers. To maximize this clinical advantage, it is highly desirable to have a global salivary analyte stabilizer for proteins, RNA and DNA at ambient temperature.


METHODS
Whole saliva, saliva supernatant and saliva filtrate (5.0 microm) were treated with RPS at room temperature (RT) for up to 6 days and then subjected to SDS-PAGE. Immunoblotting of beta-actin and cystatin C were used to evaluate protein stability. For salivary DNA/RNA, whole saliva was incubated with RPS at RT for up to 10 weeks. After extracting total DNA/RNA in samples at week 0, 2, 6 and 10, DNA stability was assayed by chromosome 18 DNA qPCR and RNA stability by beta-actin mRNA RT-qPCR.


RESULTS
beta-actin completely degraded in all types of saliva samples after 6-day incubation at RT. However, 24.0%, 91.4% and 89.3% of beta-actin remained intact with RPS for whole saliva, saliva supernatant and filtrate, respectively. Similarly, 70.3% of cystatin C in supernatant remained intact in the presence of RPS. For salivary DNA/RNA, the cycle threshold (Ct) values showed no significant change for chromosome 18 DNA and beta-actin mRNA in RPS-incubated saliva during the 10-week time course while significant increase in Ct values were observed in controls without RPS for both beta-actin mRNA and DNA.


CONCLUSIONS
RPS provided effective concurrent stabilization to salivary DNA/RNA in whole saliva for up to 10 weeks and proteins in saliva filtrate for 6 days at RT. We also achieved separation of saliva supernatant from cellular elements by a simple filtration step (bypassing the need for centrifugation).
Jiang
Jiang
J
Dental Research Institute, UCLA School of Dentistry, Los Angeles, CA 90095, USA.
Park
Noh Jin
NJ
Hu
Shen
S
Wong
David T
DT
D000328:Adult; D014408:Biomarkers, Tumor / Q000737:chemistry*; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D055088:Early Detection of Cancer; D006801:Humans; D009062:Mouth Neoplasms / Q000175:diagnosis; D012313:RNA / Q000737:chemistry*; D012463:Saliva / Q000378:metabolism; D012471:Salivary Proteins and Peptides / Q000737:chemistry*; D012680:Sensitivity and Specificity; D013696:Temperature
D014408:Biomarkers, Tumor; D012471:Salivary Proteins and Peptides; D012313:RNA; D004247:DNA