High resolution of microRNA signatures in human whole saliva

Abstract

Objective: Identifying discriminatory human salivary RNA biomarkers reflective of disease in a low-cost non-invasive screening assay is crucial to salivary diagnostics. Recent studies have reported both mRNA and microRNA (miRNA) in saliva, but little information has been documented on the quality and yield of RNA collected. Therefore, the aim of the present study was to develop an improved RNA isolation method from saliva and to identify major miRNA species in human whole saliva.

Design: RNA samples were isolated from normal human saliva using a combined protocol based on the Oragene RNA collection kit and the mirVana miRNA isolation kit in tandem. RNA samples were analysed for quality and subjected to miRNA array analysis.

Results: RNA samples isolated from twenty healthy donors ranged from 2.59 to 29.4 μg/ml saliva and with 1.92-2.16OD(260/280 nm) ratios. RNA yield and concentration of saliva samples were observed to be stable over 48 h at room temperature. Analysis of total salivary RNA isolated from these twenty donors showed no statistical significance between sexes; however, the presence of high-, medium-, and low-yield salivary RNA producers was detected. MiRNA array analysis of salivary RNA detected five abundantly expressed miRNAs, miR-223, miR-191, miR-16, miR-203, and miR-24, that were similarly described in other published reports. Additionally, many previously undetected miRNAs were also identified.

Conclusion: High quality miRNAs can be isolated from saliva using available commercial kits, and in future studies, the availability of this isolation protocol may allow specific changes in their levels to be measured accurately in various relevant diseases.

Figure 1

Stabilization of saliva for RNA isolation. Incubation of whole saliva with Oragene^®•RNA solution stabilized total RNA for two days at room temperature. (A) Bioanalyzer data demonstrating the total RNA extracted from whole saliva at Day 0 from three donors with total RNA yield per ml of saliva and OD_260/280 ratios indicated. (B) Bioanalyzer data of total RNA extracted from the same stabilized whole saliva samples two days later.

Figure 2

The variable RNA concentration in the saliva from donors is a result of the bacterial contribution and not fluctuations in mammalian RNA. (A) Graphic representation of the RNA concentrations (in 50 μl eluted volume) normalized to 1 ml of whole saliva obtained from four donors over the course of three days. (B) The total bacterial 16S rRNA contribution across the salivary samples in the four donors over three days. (C) The mean C_T values ± standard error of snU6, 18S, GAPDH, and miRNA let-7b plotted for each of the four donors across three days. Data shown were obtained from three replicates.

Figure 3

Relatively high RNA yields were collected from the saliva of 20 healthy donors. (A) Scatter plot analysis of RNA concentration in samples purified from 1 ml of whole saliva from males compared to females. High, medium, and low yield producers are approximately defined as >400, 200–400, and <200 ng/μl, respectively. (B) Linear regression analysis of RNA collected from donors using two different operators. Data shown were obtained from the same starting biological samples.