Method: Cultured normal (OKF6) and dysplastic (DOK) oral keratinocytes were treated twice-daily with a solution of alcohol-containing mouthwash (ACM) or non-alcoholic mouthwash (NAM) from one of two parent brands (A-ACM, A-NAM, B-ACM, B-NAM). After 1 week, RNA was extracted and the mRNA transcriptomes were sequenced with Ion Proton™. Unexposed cells were also collected and RNA extracted at Day 0 as a control. Sequencing data was then analysed through hierarchical gene clustering, principal component analysis and differential expression analysis.
Result: Analysis of raw data showed good quality reads with an average call error of 0.398% and an average of 90% of reads mapping to the human reference genome. Hierarchical clustering of highly expressed genes and principal component analysis revealed the most significant clustering occurred according to the origin of the cell line (normal vs dysplastic), indicating distinctly different transcriptomes. Secondary clustering was noted corresponding to exposure length (Day 0 vs Day 7). The greatest number of differentially expressed genes were noted between the Day 0 and Day 7 samples, followed by differences in expression between the “A-brand” samples to “B-brand” samples.
Conclusion: Overall, the number of repeated exposures was the tested variable found to have the greatest effect on differences in the mRNA transcriptome and gene expression profile, followed by brand of mouthwash. The high quality and versatility of the produced data supports the use of next-generation sequencing as a valid investigative tool to evaluate changes in the gene expression profile of oral keratinocytes.