Optimized liquid-based cytology for the cellular and molecular analysis of oral keratinocytes: A promising diagnostic tool.

BACKGROUND

Liquid-based cytology (LBC) has improved exfoliative cytology by facilitating the extraction of more precise information from epithelial cells. The aim of this study was to optimize a protocol using a conventional cytobrush to perform LBC, obtaining oral keratinocytes for their further cellular and molecular analysis.

METHODS

LBC was performed in 30 healthy donors from buccal mucosa. We evaluated the use of diethyl pyrocarbonate (DEPC)-treated Dulbecco's Modified Eagle Medium (DMEM) medium right after the collection of the cells. Cell morphology and viability were determined by Orcein staining and flow cytometry, respectively. RNA was extracted by the trizol method, and evaluated with spectrometry and electrophoresis. Finally, RNA was copied into cDNA and GAPDH and TLR2 genes were amplified by reverse transcription polymerase chain reaction (RT-PCR) and quantitative reverse transcription polymerase chain reaction (RT-qPCR) using specific primers.

RESULTS

Only DEPC-treated DMEM preserved the viability of intact intraepithelial keratinocytes. RNA quantity and quality improves in samples treated with DEPC. RNA integrity is comparable with a cell line control. GAPDH gene was successfully amplified by RT-PCR and RT-qPCR.

CONCLUSIONS

Therefore, LBC performed under these conditions becomes a reproducible technique for the retrieval of intraepithelial oral keratinocytes with good cell viability for cytomorphometric analysis, and extraction of good RNA quality suitable for molecular analyses such as PCR. We propose this LBC protocol as a complementary method to the cellular and molecular study of oral mucosa pathologies; however, it requires further study.

© 2020 Wiley Periodicals LLC.