Article: Validity of messenger RNA expression analyses of human saliva.
Clinical cancer research : an official journal of the American Association for Cancer Research
2006 Volume 12, Issue 17, Page(s) 5033–5039
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 ... ...
| 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, 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. |
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| MeSH term(s) | Actins/genetics ; Gene Expression Profiling ; Glyceraldehyde-3-Phosphate Dehydrogenases/genetics ; Humans ; Oligonucleotide Array Sequence Analysis/methods ; RNA, Messenger/genetics ; Reproducibility of Results ; Reverse Transcriptase Polymerase Chain Reaction/methods ; Ribosomal Proteins/genetics ; Saliva/chemistry |
| Chemical Substances | Actins ; RNA, Messenger ; Ribosomal Proteins ; ribosomal protein P0 ; Glyceraldehyde-3-Phosphate Dehydrogenases (EC 1.2.1.-) |
| Language | English |
| Publishing date | 2006-09-01 |
| Publishing country | United States |
| Document type | Journal Article ; Research Support, N.I.H., Extramural |
| ZDB-ID | 1225457-5 |
| ISSN | 1557-3265 ; 1078-0432 |
| ISSN (online) | 1557-3265 |
| ISSN | 1078-0432 |
| DOI | 10.1158/1078-0432.CCR-06-0501 |
| Database | MEDical Literature Analysis and Retrieval System OnLINE |
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