Abstract
BackgroundGene expression changes resulting from conditions such as disease, environmental stimuli, and drug use, can be monitored in the blood. However, a less invasive method of sample collection is of interest because of the discomfort and specialized personnel necessary for blood sampling especially if multiple samples are being collected. Buccal mucosa cells are easily collected and may be an alternative sample material for biomarker testing. A limited number of studies, primarily in the smoker/oral cancer literature, address this tissue's efficacy as an RNA source for expression analysis. The current study was undertaken to determine if total RNA isolated from buccal mucosa could be used as an alternative tissue source to assay relative gene expression.MethodsTotal RNA was isolated from swabs, reverse transcribed and amplified. The amplified cDNA was used in RT-qPCR and microarray analyses to evaluate gene expression in buccal cells. Initially, RT-qPCR was used to assess relative transcript levels of four genes from whole blood and buccal cells collected from the same seven individuals, concurrently. Second, buccal cell RNA was used for microarray-based differential gene expression studies by comparing gene expression between a group of female smokers and nonsmokers.ResultsAn amplification protocol allowed use of less buccal cell total RNA (50 ng) than had been reported previously with human microarrays. Total RNA isolated from buccal cells was degraded but was of sufficient quality to be used with RT-qPCR to detect expression of specific genes. We report here the finding of a small number of statistically significant differentially expressed genes between smokers and nonsmokers, using buccal cells as starting material. Gene Set Enrichment Analysis confirmed that these genes had a similar expression pattern to results from another study.ConclusionsOur results suggest that despite a high degree of degradation, RNA from buccal cells from cheek mucosa could be used to detect differential gene expression between smokers and nonsmokers. However the RNA degradation, increase in sample variability and microarray failure rate show that buccal samples should be used with caution as source material in expression studies.
Highlights
Gene expression changes resulting from conditions such as disease, environmental stimuli, and drug use, can be monitored in the blood
Buccal RNA samples were found to be severely degraded with RNA Integrity Numbers (RINs) routinely less than three
Results shown are the average value from three replicates for each primer pair as input material from each subject. * Primers used in qPCR are given by gene heading followed by 5’ position of amplification on mRNA
Summary
Gene expression changes resulting from conditions such as disease, environmental stimuli, and drug use, can be monitored in the blood. Buccal mucosa cells are collected and may be an alternative sample material for biomarker testing. For studies performed in human subjects, a less invasive tissue source for biomarker monitoring is of interest due to the discomfort, required skill level, and cost of blood collection, especially for repeated-measures studies. QPCR has been used to detect expression changes in genes from the P450 family using snap frozen surgical buccal plug samples [3] and from brushed exfoliated buccal cells [4,5] These studies suggested that buccal cells might serve as an alternative to blood in qPCR assays examining gene expression profiles after exposure to environmental toxins, tobacco smoke, drugs, nutrients, or the presence of certain cancers. A successful study of this type would more clearly suggest that buccal cells have efficacy as source material for biomarker discovery or in a gene expression monitoring system than earlier studies
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