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Letters to the Editor |
UCLA1
Dental Research Institute
2 School of Dentistry
3 Johnson Comprehensive Cancer Center
4 Division of Head and Neck Surgery/Otolaryngology
5 Henry Samueli School of Engineering
6 Molecular Biology Institute, University of California at Los Angeles, CA
aAddress Correspondence to this author at: UCLA School of Dentistry, Dental Research Institute, 73–017 CHS 10833 Le Conte Avenue, Los Angeles, CA 90095. Fax 310-825-7609; email dtww{at}ucla.edu.
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 ß-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 a 12% decrease and a 4% increase at day 7 compared with day 0. Consistent with the RT-qPCR data, the RL sample showed the biggest decrease, with 47% and 0% increases at day 7 and day 0, respectively. The SI sample showed a 6% decrease and a 23% increase. RPS showed the minimum change in mRNA, with a 3% decrease and <1% increase [data not shown but were in part published in (3)]. The decrease in mRNA signal is likely due to mRNA degradation, and the increase in the signal may be due to gene induction in desquamated oral epithelial cells in the saliva. Such a gene induction phenomenon has been observed in whole blood incubated at room temperature(4). Together these data suggest that RPS can best stabilize salivary mRNAs at room temperature for up to 7 days with minimum change in RNA level and complexity.
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To further test the stability of salivary RNA in RPS-incubated samples, we performed an extensive time course analysis at room temperature for up to 12 weeks. We used saliva without any preservation, incubated for the same periods of time as a control. The results of RT-qPCR for ß-actin mRNA are shown in Fig. 1B
. The control CT values increased after 2 weeks and remained high until the end of the time course. In the presence of RPS, however, there was no increase in the CT values for up to 12 weeks. Throughout the time course, we detected significant differences in CT values between these 2 sets of samples (overall P <0.001) at all time points. We observed similar RT-qPCR results for interleukin-8 mRNA (Fig. 1C
). Together these data suggest that RPS can stabilize salivary RNA at room temperature for up to 12 weeks without significant degradation.
The ability to promptly stabilize and preserve salivary RNA on collection at room temperature is of particular clinical and practical relevance. Room-temperature compatibility allows sample collection, stabilization, and transportation without specialized conditions (4 °C, –20 °C or –80 °C), permitting saliva samples to be collected in RPS anywhere in the world at ambient temperatures and mailed into a central reference facility for analyses. This capability is an important first step toward the use of saliva as a clinical biofluid for screening diseases such as oral cancer.
In conclusion, our data demonstrate that RPS reagent can promptly stabilize the RNA in saliva at room temperature. This finding is important for the clinical application of salivary RNA as a diagnostic analyte.
Acknowledgments
We thank members of QIAGEN GmbH, Drs. Birgit Jostes, Susanne Ullmann, and Vera Hollaender for in depth discussion of this project and providing the reagents. We thank Nisa Pungpravat for critical review of the manuscript. This work was supported by NIH T32 training grant DE07296 to N.J.P. and NIH grants RO1 DE15970, UO1 DE15018, UO1 DE17790 and UO1 16275 to D.T.W.
References
The following articles in journals at HighWire Press have cited this article:
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  N. J. Park, H. Zhou, D. Elashoff, B. S. Henson, D. A. Kastratovic, E. Abemayor, and D. T. Wong Salivary microRNA: Discovery, Characterization, and Clinical Utility for Oral Cancer Detection Clin. Cancer Res., September 1, 2009; 15(17): 5473 - 5477. [Abstract] [Full Text] [PDF]
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 Z. Hu, B. G. Zimmermann, H. Zhou, J. Wang, B. S. Henson, W. Yu, D. Elashoff, G. Krupp, and D. T. Wong Exon-Level Expression Profiling: A Comprehensive Transcriptome Analysis of Oral Fluids Clin. Chem., May 1, 2008; 54(5): 824 - 832. [Abstract] [Full Text] [PDF]
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D. T. Wong Salivary Transcriptome Clin. Cancer Res., February 15, 2007; 13(4): 1350 - 1350. [Full Text] [PDF]
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