RNAprotect saliva: An optimal room- temperature stabilization reagent for the salivary transcriptome.

To the Editor: Quantitative analysis can now be performed on a panel of human salivary mRNAs identified as potential markers for oral cancer (1, 2). Translational and clinical applications of salivary transcriptome diagnostics require RNA degradation in saliva to be stopped at the time of collection and until analysis, preferably with room-temperature–compliant stabilization reagents. We compared 3 RNA stabilizing reagents for their abilities to stabilize salivary RNA at room temperature: SUPERase InTM RNase Inhibitor (SI), RNALater® (RL), and RNAprotect® Saliva Reagent (RPS). We incubated saliva samples with these reagents and control saliva samples with no stabilization reagent for up to 7 days at room temperature. We isolated RNA with the RNeasy Micro Kit (QIAGEN Inc.), and measured the amount of salivary -actin mRNA with reverse-transcriptase quantitative PCR (RT-qPCR). The cycle threshold (CT) values increased rapidly after 1 h for control and SI samples (Fig. 1A). The RL sample also exhibited an increase in CT value starting at day 1, and its CT value at day 7 was the highest of all the samples. RPS saliva samples, unlike the SI, RL, or control samples, did not show a significant increase in the CT value for up to 7 days. From day 1 through day 7, the CT values for the RPS saliva samples were statistically lower than those of the other samples (P 0.001). To further show that RPS can preserve the global level of salivary mRNAs, we preserved saliva samples with different reagents and measured mRNA by microarray (Affymetrix, U133 plus 2.0) analysis on day 0 and day 7. A robust multiarray average (RMA) program was used to find the mRNAs for which expression had decreased or increased by 4-fold at day 7 compared with day 0. We focused on mRNAs for which the RMA expression index number was 3. The control sample showed Fig. 1. RPS can stabilize salivary mRNAs for up to 12 weeks at room temperature.