Clinical examination and histopathological studies are classical methods for diagnosis of potentially malignant disorders and malignant lesions of oral cavity. Exfoliative cytology has been proposed as a complementary method, since it can provide information of epithelial cells (1).
The extraction and purification of high quality RNA is an important step in such studies. Gene expression of oral cells from smears has already been studied (3, 4). However, to our knowledge, RNA extraction methods to obtain samples for molecular analysis of oral cytology samples have not yet been used, although Pandit et al. studied exfoliated cells via saliva collection in 2013 (5). Therefore, the aim of this study was to compare three methods of oral cytology samples RNA extraction, considering concentration and purity to establish the best technique for molecular tests of oral lesions such as real-time reverse transcriptase reaction (qPCR).
MATERIAL & METHODS
Three samples of cells were taken from buccal mucosa of 30 subjects aged 30-50 years, with no visible clinical changes at the site of collection, from the Oral Medicine, Oral Surgery and Implantology Unit of Faculty of Medicine and Dentistry, University of Santiago de Compostela; from the Smoking Cessation Program of Heart Institute, University of Săo Paulo School and from the outpatient clinic of Oral Medicine of Institute of Science and Technology, UNESP - Univ Estadual Paulista. This study was approved by the Ethics Committee in Research of Galicia and Research Ethics Committee of Institute of Science and Technology, UNESP - Univ Estadual Paulista. Informed written consent was obtained from the subjects after they had been fully explained the nature of procedures.
Smears were collected using Orcellex Rovers Brush® (Rovers Medical Devices, NL, Holanda), without use of mouthwashes (6). The Rovers® Orcellex® Brush was placed perpendicular to the area and pressed with a firm contact into the lining mucosa. It was rotated through twenty complete turns for each site to be sampled, following the manufacturer's recommendations. In each patient, this procedure was carried out in three different places of the right buccal mucosa, and placed in a different bottle for three different analyses. A total amount of cells was collected by sampling, measured in Neubauer Chamber and it amounted to 106. In this type of sample, the intermediate and superficial cells with rare or null appearance of basal and parabasal cells are most frequently observed. Generally, anucleate cells appear. In order to ensure that nucleated cells of the intermediate layers were collected in larger quantities, twenty rotations of the cytobrush were performed at buccal mucosa collecting sites. The samples were transported in DNase, RNase and pyrogenic free tubes, protected from light, containing 2ml of Roswell Park Memorial Institute medium (RPMI) (3, 4) (Microvet, Madrid, Spain) and stored at -80°C, until RNA were obtained. The samples were frozen immediately after collection, which minimizes the RNA degradation. Furthermore, some tests were previously made to compare the RPMI with specific solutions to RNA stabilization and these results showed the same performance of RNAlater®. Also, the samples were frozen since collecting was not all made on the same day; in order to limit a degradation of RNA, the samples were collected before.
Three techniques were used for RNA total extraction from exfoliative cytology. 30 samples were extracted by Trizol® technique (Trizol® group) (Life Technologies, Madrid, Spain), 30 samples were collected using the Direct-zolTM RNA Miniprep system (Zymo Research, CA, USA) (Direct-zol® TM group) and 30 samples were extracted using RNeasy mini kit (Qiagen, CA, USA) (RNeasy group). In each group, the smears were collected from the same 10 smokers, 10 ex-smokers and 10 nonsmokers. Since tobacco use is associated with an increase in keratinization and cell maturation changes (6), the authors believed that it could alter the RNA amount.
All procedures were performed according to manufacturer´s recommendation. For Trizol® group extraction, the samples were centrifuged and incubated with Trizol® after homogenization. 200µL of chloroform was added, homogenized and centrifuged. The aqueous phase was transferred to a new tube, avoiding contact with the interface; 500µL of 100% isopropyl alcohol was added to it, homogenized by inversion and centrifuged. The supernatant was discarded without disturbing the pellet. 1mL of 75% ethanol was added to it, homogenized and centrifuged. The supernatant was removed and the pellet dried and 20µL of diethylpyrocarbonate (DEPC)-treated nuclease-free water (Life Technologies, Madrid, Spain) was added to it. The total extracted amount of RNA (1 µg) was subsequently treated with DNase I (Ambion, Inc., Carlsbad, CA, USA).
For the Direct-zolTM group extraction, 1ml of TRI-Reagent® was added to pellet and homogenized completely. 1ml of ethanol 100% was directly added to the solution and homogenized. The sample was added to Zymo-Spin IIC™ column with the collection tube and centrifuged. The column was moved to a new collection tube and the collection tube containing the filtrate was discarded. The RNA samples were treated by DNase. After, 400μl Direct-zol™ RNA, a prewash was added to the column, and centrifuged. Subsequently, the filtrate was discarded and this step was repeated. 700μl RNA wash buffer was added to the column and it was centrifuged. The column was carefully transferred from the collection tube into the RNase-free tube, 20μl DEPC-treated nuclease-free water was added to it (Life Technologies, Madrid, Spain) and it was centrifuged.
For RNeasy group extraction, the samples were centrifuged after removing the supernatant. 350μL of RTL buffer was added to the cell pellet and homogenized with vortex for 1 minute; 350μL of 70% ethanol was added, and this homogenate was transferred to the column with the collection tube and centrifuged, discarding the filtrate. 700μL of buffer RW1 was added to the column, centrifuged and the filtrate was discarded. 500μL of RPE buffer was added to it, centrifuged and the filtrate was discarded. Then, this step was repeated. The column was placed in a new collection tube without lid and centrifuged at maximum speed for 1 minute. The column was inserted into a tube with lid and 20μL of DEPC-treated nuclease-free water was added (Life Technologies, Madrid, Spain) and centrifuged. Total RNA extracted (1 µg) was treated with DNase I (Ambion, Inc., Carlsbad, CA, USA)
One microliter of RNA was used to measure the absorbance at 260 (A260) and 280 (A280) in nanometer scale (nm) in NanoDrop 1000 Spectrophotometer for each sample (Thermo Scientific, Wilmington, Delaware). The estimated RNA concentration was obtained by multiplying by 40 the value of A260 (ng/mL). The purity, which indicates the quality of RNA, was assessed by the A260/A280 and A260/A230 ratios, where A260/A280 ratio values between 1.8 and 2.0 and A260 / A230 ratio values close to 1.7 suggest RNA free of contamination. An appropriate blank solution was used to zero the spectrophotometer. Integrity was checked by electrophoresis, performed using 1% agarose gel.
Statistical analyzes of the data were performed using GraphPad Prism 5.03 software and the data were presented by median, mean and standard deviation. The significance between two groups was determined, using SPSS (v.20.0), with the Student's t and ANOVA test; p value, p<0.05 was considered to indicate a statistically significant difference. The Bonferroni test was used to obtain the optimal fit between the variables
Concentration average, standard deviation and range obtained in each technique used may be observed in Table 1 along with average, standard deviation and maximum variation of A260/A280 and A260/A230 ratios.
Samples were collected from 24 men and 6 women. Purity (A260/A280 ratio) obtained in Direct-zolTM group was higher in male subjects (average = 1.90, SD= 0.064) than in female subjects (average = 1.73; SD= 0.42) (t = 2.065, p = 0.048). There were no differences between other variables in each group.
The tobacco variable was measured using ANOVA and the distribution is statistically significant for quantitative variable RNA concentration in Trizol® group (F = 8.983, p = 0.001) and in Direct-zolTM group (F = 4.06, p = 0.029). Concentration in each subgroup can be observed in Table 2.
To achieve the optimal adjustment of variables, the tobacco smokers variables were considered as tobacco smokers (smokers now and ex-smokers) and nonsmokers (persons who have never smoked and those who smoked at one time and subsequently quit). In this case, A260/A280 ratio in RNeasy group was higher in non-smokers (average= 1.96; SD=0.06), than in smokers (1.90± 0.09; F = 4.507, p = 0.043).
Oral exfoliative cytology includes the study and interpretation of the features cells exfoliated from the oral mucosa. It was first developed by Papanicolaou and Traut (1941) (7) who studied the cells from precancerous and cancerous lesions of the cervical mucosa in an attempt to diagnose cervical neoplasia. After that, oral cytology was used for the diagnosis of oral cavity diseases (8). The cytological samples can be studied by taking different approaches but the researchers should always make efforts to understand the tissue changes (3, 9, 10), by qPCR.
The cytological smears are easily collected. They are practical and low cost (2) and may provide information about epithelial cells (1) since the cells of more superficial layers of the epithelium store information about the changes occurred during the cellular maturation process (6). Therefore, cytology may be helpful in detecting early changes in smokers and in biomonitoring (6). Thus, it can be used as a non-invasive collection technique to study genes expressions in this type of patients and other risk groups.
The aim of this study was to find the best RNA extraction technique, including minimization of time and cost to obtain samples of oral lesions utilized in qPCR.
Real-time technology (qPCR) has significantly extended the use and scope of PCR assays (11) and it is considered the gold standard technique to study transcript levels of a specific set of genes. Therefore, qPCR is a technique with high demand that has to assure high reliability, sensitivity and reproducibility (12). RNA quantification of the extracted samples is considered an important step (13), because it is necessary to use the same RNA amounts in an analysis when comparing different samples. Naturally, it should be accurately quantified.
It is important to note that the RNA sample obtained by cytology is partially degraded and it creates low-levels of transcripts detection (13). Furthermore, significant amounts of RNases are present in the saliva (14) and RNA degrades markedly in vivo, owing to the natural regulation of mRNAs in response to environmental stimuli (13). Therefore, the process should be executed carefully to minimize sample degradation, before extraction and analysis (5).
The pH and ionic strength of the solutions used for spectrophotometric analysis can influence substantially the qualitative and quantitative determinations of nucleic acids. For example, the RNA solubilized water can alter the A260/280 ratio of the same RNA preparation (15). In this study, DEPC-treated nuclease-free water to solubilized RNA was used in all techniques.
The highest average concentration was observed in the Trizol® group, followed by the Direct-zolTM group. The lowest average concentration in RNeasy group was observed. Trizol® solubilization and extraction method was developed as a general method for deproteinizing of the RNA. This method is particularly advantageous in situations where cells or tissues have a considerable amount of endogenous RNases or when the separation of cytoplasmic RNA from nuclear RNA is unpractical (16).
Trizol® is a monophasic solution of phenol and guanidinium isothiocyanate which, simultaneously, solubilizes biological material and denatures the protein. After the solubilization, the addition of chloroform causes the phase separation, where protein is extracted to the organic phase, DNA resolves at the interface, and RNA remains in the aqueous phase (16).
According to the manufacturer, Direct-zolTM RNA Miniprep system is a quick spin column purification of high-quality total RNA directly from Trizol® and it can extract RNA purified from human epithelial cells. In this study, an average amount of RNA concentration suitable for PCR studies was observed and RNA samples are of high quality.
According to the manufacturer, the RNeasy Kit is designed to purify RNA from small amounts of starting material. This kit has been used in studies of qPCR (3, 4). However, the low amount of total RNA obtained hinders the implementation of studies of several genes in the same sample.
The ratio of absorbance at 260 and 280nm (the A260/280 ratio) is frequently used to assess the purity of RNA and DNA preparations. The maximum absorbance for nucleic acids and proteins is 260 and 280 nm, respectively. The A260/A280 ratio has been used as a measure of purity. Generally, it is accepted as “pure” for RNA the ratio about 2.0. Similarly, another contamination absorbance has 230nm and the A260/A230 ratio values for “pure” are in the range of 2.0-2.2 . In this study, the average A260/A280 ratio observed in the RNeasy group was very close to 2 (1.939), but the average of Direct-zoLTM and Trizol® groups was lower: 1.8693 and 1.77, respectively. The average A260/A230 ratio observed in the Direct-zolTM group was the nearest to 2 (1.69), and the average of the Trizol® and RNeasy group was lower: 0.75 and 0.166, respectively. Therefore, it was observed that the RNA Direct-zolTM group has the highest purity, followed by RNeasy and Trizol® groups, considering the two ratios.
The Direct-zolTM RNA Miniprep system and RNeasy minikit are methods which use column purification to eliminate waste and contaminants through precipitation using alcohol in the silica that is inside the column. This may be a limitation, since much cytology debris may obstruct the membrane either partially or completely. On the other hand, the Trizol® method has certain limitations, since it depends greatly on the pipetting due to the aqueous phase when it is directly removed by the operator.
One of the advantages of Direct-zolTM kit is the inclusion of the treatment with DNAse. It is important to note that the RNA sample has been treated with DNase to avoid genomic-DNA contamination (13).
Furthermore, we would like to highlight some findings: RNA concentration was higher in non-smoking and ex-smokers than smokers. Higher keratinization of buccal mucosa of smokers (17) may be related to RNA lower concentration found in the three methods which were evaluated.
Considering that A260/A280 ratio was higher in non-smokers than in smokers belonging to RNeasy group, further studies to clarify these findings are needed.
According to the manufacturer, the Trizol reagent used in the kits Trizol® and Direct-zolTM is mainly composed of phenol. It may be toxic if swallowed. In contact with skin or if inhaled it may cause severe skin burns and eye damage, respiratory irritation. Besides, it is suspected of causing genetic defects, may cause damage to organs through prolonged or repeated exposure and is harmful to aquatic life with long lasting effects. It is noteworthy that it should be used carefully and disposed properly. Personal protective equipment is recommended. The RNeasy minikit does not contain phenol.
This is a preliminary study and additional studies are necessary to give more information about the quality of the samples. Direct-zolTM group had the highest purity, followed by RNeasy and Trizol® groups, considering the two ratios used. Considering the aspects such as concentration, purity and time spent in the procedures, Direct-zol® group obtained the best results. Moreover, this kit can be easily implemented, since its use does not require any special training. Finally, Trizol® is a lower- cost option.