Exon-level expression profiling: A comprehensive transcriptome analysis of oral fluids

BACKGROUND: The application of global gene expression profiling to saliva samples is hampered by the presence of partially fragmented and degraded RNAs that are difficult to amplify and detect with the prevailing technologies. Moreover, the often limited volume of saliva samples is a challenge to quantitative PCR (qPCR) validation of multiple candidates. The aim of this study was to provide proof-of-concept data on the combination of a universal mRNA-amplification method with exonarrays for candidate selection and a multiplex preamplification method for easy validation. METHODS: We used a universal ITIRNA-specific linear-amplification strategy in combination with Affyrnetrix Exon Arrays to amplify salivary RNA from 18 healthy individuals on the nanogram scale. Multiple selected candidates were preamplified in one multiplex reverse transcription PCR reaction, cleaned up enzymatically, and validated by qPCR. RESULTS: We defined a salivary exon core transcriptorne (SECT) containing 851 transcripts of genes that have highly similar expression profiles in healthy individuals. A subset of the SECT transcripts was verified by qPCR analysis. Informatics analysis of the SECT revealed several functional clusters and sequence motifs. Sex-specific salivary exon biomarkers were identified and validated in tests with samples from healthy individuals. CONCLUSIONS: It is feasible to use samples containing fragmented RNAs to conduct high-resolution expression profiling with coverage of the entire transcriptorne and to validate multiple targets from limited amounts of sample. (c) 2008 American Association for Clinical. Chemistry.