Validity of messenger RNA expression analyses of human saliva.
OBJECTIVE
The origins of expression microarray and reverse transcription-PCR (RT-PCR) signals in human saliva were evaluated.


METHODS
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, beta-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, beta-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, beta-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.
Kumar
Shalini V
SV
Laboratory of Human Toxicology and Molecular Epidemiology, Wadsworth Center, New York State Department of Health, Albany, New York 12201-0509, USA.
Hurteau
Gregory J
GJ
Spivack
Simon D
SD
D000199:Actins / Q000235:genetics; D020869:Gene Expression Profiling; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics; D006801:Humans; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods; D012333:RNA, Messenger / Q000235:genetics*; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D012269:Ribosomal Proteins / Q000235:genetics; D012463:Saliva / Q000737:chemistry*
D000199:Actins; D012333:RNA, Messenger; D012269:Ribosomal Proteins; C054675:ribosomal protein P0; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases