Validity of Messenger RNA Expression Analyses of Human Saliva

Abstract

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, h-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, h-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, h-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-RTsaliva 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.Large-scale human cancer studies for the assessment of functional genomics or gene-environment interactions require a reliable source of RNA that can be accessed with minimal invasiveness, and is compatible with downstream, highthroughput applications. Options that have emerged include brush-exfoliated buccal cells (1), urine (2), and saliva (3, 4). Given the potential translational importance of mRNA expression biomarkers in such noninvasively acquired biospecimens, it is imperative that the RNA specificity of gene expression studies from these specimens be confirmed.Many human transcripts have homologous sequences in nontranscribed genomic DNA sequences, remote from the structurally active coding genes, which are termed pseudogenes. Common examples include h-actin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), acidic ribosomal phosphoprotein P0 (36b4), hypoxanthine phosphoribosyl transferase, glutathione S-transferases M1 and P1, and glutathione peroxidase. These pseudogenes are intronless, often have poly(A) tails, and have extremely high homology wit...

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