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Clinical Chemistry 54: 824-832, 2008. First published March 20, 2008; 10.1373/clinchem.2007.096164
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(Clinical Chemistry. 2008;54:824-832.)
© 2008 American Association for Clinical Chemistry, Inc.

Molecular Diagnostics and Genetics

Exon-Level Expression Profiling: A Comprehensive Transcriptome Analysis of Oral Fluids

Zhanzhi Hu1,4, Bernhard G. Zimmermann1,4, Hui Zhou1, Jianghua Wang1, Bradley S. Henson1, Weixia Yu1, David Elashoff2, Guido Krupp7 and David T. Wong1,3,4,5,6,a

1 Dental Research Institute, 2 Department of Biostatistics, 3 School of Dentistry, 4 Jonsson Comprehensive Cancer Center, 5 Division of Head and Neck Surgery/Otolaryngology, and 6 Henry Samueli School of Engineering, University of California, Los Angeles, CA; 7 AmpTec GmbH, Hamburg, Germany.

aAddress correspondence to this author at: Dental Research Institute, 73-017 Center for Health Sciences, 10833 Le Conte Ave., University of California, Los Angeles, CA 90095-1668. e-mail dtww{at}ucla.edu.

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 exon arrays for candidate selection and a multiplex preamplification method for easy validation.

Methods: We used a universal mRNA–specific linear-amplification strategy in combination with Affymetrix 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 transcriptome (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 transcriptome and to validate multiple targets from limited amounts of sample.






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Copyright © 2008 by the American Association for Clinical Chemistry.