Comparison of RNA Extraction Methods for Molecular Analysis of Oral Cytology

Mônica Ghislaine Oliveira Alves; Mario Pérez-Sayáns; Maria-Elena Padín-Iruegas; Maria Dolores Reboiras-López; José Manuel Suarez-Peńaranda; Rafael López-López; Celina Faig Lima Carta; Jaqueline Scholz Issa; Abel García-García; Janete Dias Almeida

doi:10.15644/asc50/2/2

Comparison of RNA Extraction Methods for Molecular Analysis of Oral Cytology

Acta Stomatol Croat. 2016 Jun;50(2):108-115. doi: 10.15644/asc50/2/2.

Affiliations

¹ Department of Biosciences and Oral Diagnosis, Institute of Science and Technology, UNESP - Univ Estadual Paulista, Săo José dos Campos, Săo Paulo, Brazil.; School of Dentistry, Universidade Braz Cubas, Mogi das Cruzes, Brazil.
² Oral Medicine, Oral Surgery and Implantology Unit, Faculty of Medicine and Dentistry, Santiago de Compostela, Spain .
³ Human Anatomy and Embriology Area; Functional Biology and Health Sciences Department. University of Vigo, Pontevedra, Spain .
⁴ Department of Pathology, Forensic Sciences, University Hospital and School of Medicine of Santiago de Compostela, Santiago de Compostela, La Coruńa Spain .
⁵ Department of Medical Oncology Hospital Clinico Universitario of Santiago de Compostela, La Coruńa, Spain .
⁶ Department of Biosciences and Oral Diagnosis, Institute of Science and Technology, UNESP - Univ Estadual Paulista, Săo José dos Campos, Săo Paulo, Brazil .
⁷ Smoking Cessation Program, Area of Cardiology, Heart Institute, University of Săo Paulo, School of Medicine, Hospital das Clínicas, Săo Paulo, Brazil.

Abstract

Objective of work: The aim of this study was to compare three methods of RNA extraction for molecular analysis of oral cytology to establish the best technique, considering its concentration and purity for molecular tests of oral lesions such as real-time reverse transcriptase reaction.

Material and methods: The sample included exfoliative cytology from the oral cavity mucosa of patients with no visible clinical changes, using Orcellex Rovers Brush^®. The extraction of total RNA was performed using the following three techniques: 30 samples were extracted by Trizol® technique, 30 by the Direct-zol^TM RNA Miniprep system and 30 by the RNeasy mini Kit. The absorbance was measured by spectrophotometer to estimate the purity. The estimated RNA concentration was obtained by multiplying the value of A260 (ng/mL) by 40. Statistical analysis of the obtained data was performed using GraphPad Prism 5.03 software with Student t, analysis of variance and Bonferroni tests, considering p ≤0.05.

Results: Trizol® group revealed higher average concentration, followed by Direct-zol^TM and Rneasy group. It was observed that the RNA Direct-zol^TM group had the highest purity, followed by RNeasy and Trizol® groups, allowing for the two ratios.

Conclusion: Considering all aspects, concentration, purity and time spent in the procedures, the Direct-zol^TM group showed the best results.

Keywords: RNA; cytology; oral; oral exfoliative cytology.