Examining smoking-induced differential gene expression changes in buccal mucosa.
BACKGROUND
Gene 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.


METHODS
Total 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.


RESULTS
An 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.


CONCLUSIONS
Our 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.
Kupfer
Doris M
DM
Civil Aerospace Medical Institute, Federal Aviation Administration, Oklahoma City, Oklahoma, USA. doris.kupfer@faa.gov
White
Vicky L
VL
Jenkins
Marita C
MC
Burian
Dennis
D
D000328:Adult; D005260:Female; D020869:Gene Expression Profiling; D053263:Gene Regulatory Networks; D006801:Humans; D008875:Middle Aged; D009061:Mouth Mucosa / Q000378:metabolism*; D020411:Oligonucleotide Array Sequence Analysis; D012313:RNA / Q000032:analysis / Q000378:metabolism; D012907:Smoking
D012313:RNA