Characterization of RNA in saliva

Abstract

Background: We have previously shown that human mRNAs are present in saliva and can be used as biomarkers of oral cancer. In this study, we analyzed the integrity, sources, and stability of salivary RNA.

Methods: We measured the integrity of salivary RNA with reverse transcription followed by PCR (RT-PCR) or RT-quantitative PCR (RT-qPCR). To study RNA entry sites into the oral cavity, we used RT-PCR analysis of salivary RNA from the 3 major salivary glands, gingival crevice fluid, and desquamated oral epithelial cells. We measured stability of the salivary beta-actin mRNA by RT-qPCR of salivary RNA incubated at room temperature for different periods of time. We measured RNA association with other macromolecules by filtering saliva through pores of different sizes before performing RT-qPCR. To assess RNA-macromolecule interaction, we incubated saliva with Triton X-100 for different periods of time before performing RT-qPCR.

Results: In most cases, we detected partial- to full-length salivary mRNAs and smaller amounts of middle and 3' gene amplicons compared with the 5'. RNA was present in all oral fluids examined. Endogenous salivary beta-actin mRNA degraded more slowly than exogenous beta-actin mRNA, with half-lives of 12.2 and 0.4 min, respectively (P <0.001). Salivary RNA could not pass through 0.22 or 0.45 mum pores. Incubation of saliva with Triton X-100 accelerated degradation of salivary RNA.

Conclusions: Saliva harbors both full-length and partially degraded forms of mRNA. RNA enters the oral cavity from different sources, and association with macromolecules may protect salivary RNA from degradation.

Figure 1.

Integrity measurement of salivary RNA. (A), list of 5 NSCT genes, their cDNA length, and expected sizes of PCR amplicons shown in panels B and C. The 3′ and 5′ in parentheses indicate the location of amplicons on the gene. The m/3′ in parentheses for β-actin indicates that the amplification region is located between the middle and the 3′ end of the β-actin mRNA. (B), RT-PCR of salivary RNA with primers that yield short PCR amplicons. For RNA isolation, 560 μL of the supernatant phase of saliva was used. PCR was done for 40 cycles. Lanes 1–8 represent salivary RNA from 8 different participants. Lane 3* contains 5-fold more RNA starting material than lane 3, and PCR was done for 45 cycles instead of 40 cycles. Lane (–) represents a negative control, for which an equal volume of water was used during the PCR instead of the reverse transcription products. (C), salivary RNA from the same participants as in panel B was used for RT-PCR (45 cycles) that yielded long PCR amplicons. The inset indicated by (+) next to the GAPDH gel image shows the result of the positive control, in which RT-PCR was performed with RNA isolated from the MCF7 cell line. SAT product for participant 8 (lane 8) gave triplicate bands, none of which matched the expected PCR size (∗). (D and E), RT-qPCR of salivary RNA from 8 participants with β-actin (D) and IL8 (E) primers that target different regions of β-actin and IL8 mRNAs as indicated. Thick horizontal lines indicate the medians. The boxes represent the interval between the 25th and 75th percentiles. Maximum and minimum values are indicated by the vertical lines.

Figure 2.

Measurement of RNA from different oral fluids. WS, supernatant of whole saliva; SM, submandibular saliva; SL, sublingual saliva; P, parotid saliva; OEC, oral epithelial cells. (A), RT-PCR of oral fluids obtained from 6 participants (lanes 1–6) with β-actin primers. Oral fluids were collected as described in the Materials and Methods. PCR was performed for 45 cycles. (B), RNA in oral fluid samples from 1 participant was analyzed by U133 plus 2.0 expression-based microarray (Affymetrix). ▦, total number of genes present in each site; □, numbers of NSCT genes.

Figure 3.

Stability of salivary and nonsalivary RNA in saliva. (A), at time 0, 2 μg of mouse ES-LW1 cell line total RNA was added per 1 mL of the supernatant phase of human saliva, and the samples were incubated at room temperature for up to 20 min. At each time point, 300 μL of saliva was removed for RT-qPCR to measure mβ-actin mRNA. The amount of RNA quantified at each time point was normalized to the amount measured at time 0. Saliva samples from 3 participants were used. Error bars represent SD of samples from these participant tested in 1 RT-qPCR reaction. (B), the same samples as in A were used, but the qPCR was performed with hβ-actin primers to measure the hβ-actin mRNA.

Figure 4.

Stability of salivary RNA after incubation with Triton X-100. At time 0, saliva supernatant was mixed with either Triton X-100 or water (no Triton) and incubated for up to 20 min at room temperature. For RT-qPCR, salivary β-actin was measured. The amount of RNA quantified at each time point was normalized to the amount of RNA at time 0. Saliva samples from 4 participants were used. Error bars represent SD for the samples from these participants tested during 2 qPCR reactions.