Alves Mônica Ghislaine Oliveira, Pérez-Sayáns Mario, Padín-Iruegas Maria-Elena, Reboiras-López Maria Dolores, Suarez-Peńaranda José Manuel, López-López Rafael, Carta Celina Faig Lima, Issa Jaqueline Scholz, García-García Abel, Almeida Janete Dias
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.
Oral Medicine, Oral Surgery and Implantology Unit, Faculty of Medicine and Dentistry, Santiago de Compostela, Spain .
Acta Stomatol Croat. 2016 Jun;50(2):108-115. doi: 10.15644/asc50/2/2.
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.
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 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.
Trizol® group revealed higher average concentration, followed by Direct-zol and Rneasy group. It was observed that the RNA Direct-zol group had the highest purity, followed by RNeasy and Trizol® groups, allowing for the two ratios.
Considering all aspects, concentration, purity and time spent in the procedures, the Direct-zol group showed the best results.
本研究旨在比较三种用于口腔细胞学分子分析的RNA提取方法，以确定最佳技术，同时考虑其用于口腔病变分子检测（如实时逆转录反应）时的浓度和纯度。
样本包括使用Orcellex Rovers Brush从无明显临床变化的患者口腔黏膜获取的脱落细胞学样本。使用以下三种技术进行总RNA提取：30个样本采用Trizol®技术提取，30个样本采用Direct-zol RNA Miniprep系统提取，30个样本采用RNeasy mini试剂盒提取。用分光光度计测量吸光度以评估纯度。通过将A260（ng/mL）的值乘以40获得估计的RNA浓度。使用GraphPad Prism 5.03软件进行学生t检验、方差分析和Bonferroni检验对所得数据进行统计分析，以p≤0.05为标准。
Trizol®组显示出较高的平均浓度，其次是Direct-zol组和Rneasy组。观察到RNA Direct-zol组的纯度最高，其次是RNeasy组和Trizol®组，这两种方法都考虑了两个比率。
综合考虑所有方面，即浓度、纯度和操作过程中花费的时间，Direct-zol组显示出最佳结果。
