Liquid-based Cytology for Oral Keratinocytes Cellular and Molecular Study

Objectives: To validate a liquid-based cytology with a conventional cytobrush as a non-invasive method for obtaining and analysing oral keratinocytes with cellular and molecular assays.
Methods: After approval of the Institutional Ethics Committee, cytology from clinically, normal appearing oral mucosa of 15 healthy donors, was performed on the right or left cheek using a conventional cytobrush. DMEM media was used as the transport medium for cell recovery. Orcein staining was performed to verify cell morphology and Neubauer counting chamber was used to determine the number of recovered cells. Subsequently, RNA extraction was performed using the Trizol^® method. RNA concentration and purity were evaluated by spectrophotometry and electrophoresis. Finally, RNA was copied into cDNA and GAPDH gene was amplified by RT-PCR using specific primers.
Results: i) 1x10⁶ cells/ml on average were obtained ii) The keratinocytes obtained showed both plasmatic and nucleus membranes integrity. iii) The average of recovered RNA concentration was 180 ng/ul and absorbance range A260/280 was 1.6-1.9, indicating high purity. Additionally, electrophoresis in 1% agarose gel displayed RNA integrity. iv) GAPDH gene was correctly amplified.
Conclusions: Oral liquid-based cytology with a conventional cytobrush can be easily performed with less cost and discomfort to the patient. Quantity of isolated intraepithelial keratinocytes allowed the extraction of good quality RNA suitable for molecular analyses such as PCR. Although it is proposed as a complementary non-invasive method to the clinical and traditional histological evaluation for the cellular and molecular study of oral mucosa pathologies; it requires further study.