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Validity of Messenger RNA Expression Analyses of Human Saliva

Authors' Affiliations: ¹ Laboratory of Human Toxicology and Molecular Epidemiology, Wadsworth Center, New York State Department of Health, Albany, New York, ² Ordway Research Institute, ³ Pulmonary and Critical Care Medicine, Albany Medical College, Albany, New York, and ⁴ Environmental Health Sciences, School of Public Health, State University of New York, Rensselaer, New York

Requests for reprints: Simon D. Spivack, Laboratory of Human Toxicology and Molecular Epidemiology, Wadsworth Center, New York State Department of Health, P.O. Box 509, Empire State Plaza, Albany, NY 12201-0509. Phone: 518-473-0782; Fax: 518-486-1505; E-mail: spivack{at}wadsworth.org.

Purpose: The origins of expression microarray and reverse transcription-PCR (RT-PCR) signals in human saliva were evaluated.

Experimental Design: The "RNA" extracts from human saliva samples were treated with vehicle, DNase, or RNase. Two-step amplification and hybridization to Affymetrix 133A cDNA microarrays were then done. Confirmatory RT-PCR experiments used conventionally designed PCR primer pairs for the reference housekeeper transcripts encoding 36B4, ß-actin, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA sequences, which are known to be homologous to genomic DNA pseudogene sequences. Negative controls included the omission of reverse transcriptase ("no-RT") to detect any DNA-derived signal. Finally, an RNA-specific RT-PCR strategy eliminated confounding signals from contaminating genomic DNA.

Results: Microarray experiments revealed that untreated, DNase-treated, and RNase-treated "RNA" extracts from saliva all yielded negligible overall signals. Specific microarray signals for 36B4, ß-actin, and GAPDH were low, and were unaffected by RNase. Real-time quantitative RT-PCR reactions using conventional, non–RNA-specific primers on saliva samples yielded PCR products for 36B4, ß-actin, and GAPDH; DNase-treated saliva samples did not yield a PCR product, and the "no-RT" and "+RT" conditions yielded similar amounts of PCR product. The RNA-specific RT-PCR strategy, across all conditions, yielded no PCR product from saliva.

Conclusions: The combination of (a) a minimal microarray signal, which was unaffected by RNase treatment, (b) the presence of a conventional RT-PCR housekeeper product in both RNase-treated and no-RT saliva samples, (c) the absence of a conventional RT-PCR housekeeper product in DNase-treated conditions, and (d) the absence of a RNA-specific RT-PCR product shows that any microarray or RT-PCR signal in the saliva must arise from genomic DNA, not RNA. Thus, saliva extracts do not support mRNA expression studies.

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