Mônica Ghislaine Oliveira Alves1, Mario Pérez-Sayáns2, Maria-Elena Padín-Iruegas3, Maria Dolores Reboiras-López2, José Manuel Suarez-Peńaranda4, Rafael López-López5, Celina Faig Lima Carta6, Jaqueline Scholz Issa7, Abel García-García2, Janete Dias Almeida6. 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-zolTM 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-zolTM and Rneasy group. It was observed that the RNA Direct-zolTM 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-zolTM group showed the best results.
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-zolTM 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-zolTM and Rneasy group. It was observed that the RNA Direct-zolTM 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-zolTM group showed the best results.
Authors: Stephen A Bustin; Vladimir Benes; Jeremy A Garson; Jan Hellemans; Jim Huggett; Mikael Kubista; Reinhold Mueller; Tania Nolan; Michael W Pfaffl; Gregory L Shipley; Jo Vandesompele; Carl T Wittwer Journal: Clin Chem Date: 2009-02-26 Impact factor: 8.327
Authors: Mario Pérez-Sayáns; María Dolores Reboiras-López; José Manuel Somoza-Martín; Francisco Barros-Angueira; Pilar Gayoso Diz; José Manuel Gándara Rey; Abel García-García Journal: Cancer Biol Ther Date: 2010-06-25 Impact factor: 4.742
Authors: Mario Pérez-Sayáns; Abel García-García; María Dolores Reboiras-López; Pilar Gándara-Vila Journal: Int J Oncol Date: 2009-06 Impact factor: 5.650
Authors: Brady Trevisan; Alshaimaa Morsi; Julio Aleman; Martin Rodriguez; Jordan Shields; Diane Meares; Andrew M Farland; Christopher B Doering; H Trent Spencer; Anthony Atala; Aleks Skardal; Christopher D Porada; Graça Almeida-Porada Journal: Front Bioeng Biotechnol Date: 2021-03-01