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Letters to the Editor |
School of Dentistry and Dental Research Institute, School of Medicine, Jonsson Comprehensive Cancer Center and Molecular Biology Institute, University of California Los Angeles, Los Angeles, California
To the Editors:The recent article by Kumar et al. (1) in the September 1, 2006 issue of Clinical Cancer Research challenged the presence of salivary mRNA. They concluded that the source of the published RNA signals detected in human saliva is from genomic DNA contamination. This letter provides a different perspective on this subject and rebuts the investigators' conclusion.
The major discrepancy is the claim by these investigators (1) that they have reproduced the experimental conditions of the Li et al. article (2) without detecting mRNA. A careful comparison of the two articles shows that there are significant differences. First and foremost, Kumar et al. used RNAlater (Ambion Inc., Austin, TX) to stabilize salivary RNA, whereas Li et al. used Superase-In (Ambion, Inc., Austin, TX), a protein-based inhibitor efficient in inhibiting different types of nucleases, including RNase A, B, C, 1, and T1. Other than inhibiting nucleases, it has no additional effect in saliva. RNAlater, on the other hand, does a lot more than just preserving RNA. RNAlater increases cellular membrane permeability and its high salt content (ammonium sulfate) changes intracellular salt concentration, leading to leakage of cellular components, including genomic DNA. We have recently compared a number of RNA stabilizers and found RNAlater to be the least suitable, even worse than without any stabilizer (3).
Second, the methods used by the Kumar et al. cannot isolate RNA from saliva, not even from the cellular pellet. The failure to isolate RNA from the saliva cell pellet should signal concerns as it is enriched in epithelial cells and leukocytes, which often serve as an internal positive control for RNA isolation. The investigators used a different RNA isolation procedure than the one published by Li et al. (2). The failure to isolate RNA from the saliva cellular pellet suggests the inability of their procedure to harvest RNA from saliva.
The scientific credibility of the salivary transcriptome is increasing, ranging from recent studies on the characterization of salivary RNA (4), to assessing salivary RNA integrity by cDNA library analysis (5), to identifying and commercializing the most effective stabilizer, RNA Protect Saliva (QIAGEN, Valencia, CA) for research and eventual clinical applications (3). The salivary cDNA library analysis conclusively showed that none of the detected mRNA in saliva is from a pseudogene of contaminating genomic origin, independently disputing DNA as a source of templates giving rise to false-positive RNA signals (5). Other research groups, particularly from forensic sciences, are focusing on multiplex mRNA profiling for the identification of body fluids, including saliva (6–9). The most recent study from Seugnet et al. compellingly shows the value of the amylase mRNA in human saliva as a highly discriminatory biomarker for sleep drive (10).
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