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 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.
Hu
Zhanzhi
Z
Dental Research Institute, 73-017 Center for Health Sciences, University of California, Los Angeles, CA 90095-1668, USA.
Zimmermann
Bernhard G
BG
Zhou
Hui
H
Wang
Jianghua
J
Henson
Bradley S
BS
Yu
Weixia
W
Elashoff
David
D
Krupp
Guido
G
Wong
David T
DT
D005091:Exons; D005260:Female; D020869:Gene Expression Profiling; D006801:Humans; D016015:Logistic Models; D008297:Male; D020411:Oligonucleotide Array Sequence Analysis; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012463:Saliva / Q000378:metabolism*; D012732:Sex Determination Analysis
D012333:RNA, Messenger