Study population
This cross-sectional study has been carried out on randomly recruited 48 volunteer university students (24 males, 24 females) aged between 18 and 27 years. This age range was selected because it is expected that this young population group is exposed to daily mobile phone use. In short-term exposure, people are getting exposed to RF-EMR for 10, 15, 30 min or 1 h. In long-term exposure, the duration increases up to 5–6 h or even longer than this [23]. It has been reported that, on average, the person spends 90 min a day on his/her phone [2]. Written informed consent has been signed by every participant. Prior to providing the oral mucosal cells, each person was interviewed by the same researcher to standardize data collection. The specially constructed detailed questionnaire included information on age, gender, type of mobile used, frequency of mobile phone use (min/day) and the history of mobile use (number of years). The study has excluded smokers, or people who received drug therapy during the last 3 months, suffering any disease including cancer, exposed to radiotherapy including dental procedures, dietary supplements and regular mouthwash users. All subjects completed the course of the study. The research staff was ready to answer any query that may arise at any time during the study. Participants were divided into three groups based on the frequency and intensity of phone use: low mobile phone users (less than 30 min/day), medium mobile phone users (30–60 min/day) and heavy mobile phone users (more than 60 min/day). The participants were further categorized into three classes on the basis of the history of phone use: short-, medium-, and long-duration users for those who used their phones for periods shorter than 5 years, 5–10 years and longer than 10 years, respectively. Appropriate Ethical Committee approval and the informed consent of all participating subjects were obtained. It was not possible to find age-matched non-users as a control group because the wide usage of mobile phones is so profound; today each and every person uses a cell phone.
Cell samples
Participants were requested not to eat or drink 1 h prior to collecting the oral squamous epithelial cells. The oral cavity was rinsed with drinking water. Two samples were obtained, at the same time of the day; between 10 a.m. and 12 noon, from the inner surface of both sides of the donor's cheeks using sterile, small-headed plastic toothbrushes. To dislodge cells and release them, the brush was immersed in a buffered medium with repeated rotation. The collected material was centrifuged (Sigma Cold Centrifuge, Osterode am Harz, Germany) for 10 min at 14,000 rpm, and the cell pellets were harvested for RNA isolation.
Total RNA extraction
The total RNA of the oral squamous epithelial cells was extracted by TRIzol reagent (Invitrogen, California, USA) according to the manufacturer’s instructions. The cells were placed in 1.5-ml Eppendorf tubes and centrifuged at 14,000 rpm for 10 min. The pellets were washed in Dulbecco's modified Eagle medium (Euroclone group, EU) and re-centrifuged at 14,000 rpm for 10 min. The supernatant was aspirated, and 200 µl TRIzol was transferred to each tube. The cell suspensions were then vortexed for 1 min and left for incubation at room temperature (RT) for 5 min. After that, 0.2 ml chloroform/1 ml volume TRIzol was added and the tube was votexed for 30 s. The supernatant was transferred to a new tube, and 0.5 ml isopropanol and 50 μl 0.3 M sodium acetate were added to a volume of 1 ml supernatant. The contents were stored at − 20 °C for 20 min. After centrifugation at 15,000 rpm for 5 min, the resulting RNA pellets were washed with 70% ethanol. Cells were centrifuged again at 15,000 rpm at 4 °C for 3 min and washed with absolute ethanol 2 times, mildly dried and dissolved in 30 μl N.F water. The latter step was repeated, and the cells were dried for 10 min. The pellets were suspended in 30 µl N.F water. Finally, the sample’s RNA concetration was measured using Thermo Fisher Scientific NanoDrop (Vantaa, Finland), and the RNA sample was stored at − 80 °C or processed directly for cDNA synthesis.
DNase treatment protocol
The prepared RNA samples were treated with DNase using New England BioLabs DNase kit (Ipswich, Massachusetts, USA). Briefly, 10 μl of the RNA sample were mixed with 10 μl of DNase I reaction buffer 1X and 1 µl of RNase-free DNase I. The volume was completed up to 100 µl with N.F water. After mixing, the components of the mixture were incubated at 37 °C for 10 min, and then 1 µl of 0.5 M EDTA was added to the mixture and heated to 75 °C for 10 min. After cooling of the tube contents, DNA-free RNA was extracted using the protocol mentioned in the above section.
RT-PCR analysis
RNA was converted into single-stranded cDNA according to the High-Capacity cDNA Reverse Transcription Kit by Thermo Fisher Scientific (Waltham, Massachusetts, USA). A 10 µl mixture was transferred to a PCR tube along with a 10 µl of the RNA sample and incubated in Esco Swift MaxPro thermal cycler (Changi, Singapore) under the following conditions: 25 °C for 10 min, 37 °C for 10 min and 85 °C for 5 min. The cDNA sample was diluted 1:1 with F.N water and stored at − 80 °C. The PCR reaction mixture consisted of 1 µl sample cDNA and 10 µl of QuantiFast 2X Master Mix (Qiagen, Germany) including SYBR GREEN, 2 µl of the related primers and 5 µl N. F water. After mixing, the 20 µl mixtue was transferred to a PCR tube and DNA was amplified. The procedure was carried out under the following conditions: initial denaturation phase, 95 °C for 30 s, followed by 40 cycles with denaturation at 95 °C for 10 s, annealing at 60 °C for 30 s, and the extension phase was performed at 72 °C for 40 s and 72 °C for 2 min. To avoid any inter-run variation, cDNA prepared from human periodontal ligament (PDL) fibroblasts was always run together within one run. Changes in the expression of each target gene were normalized relative to the mean critical threshold (CT) values of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a housekeeping gene. The used primers in PCR description were as follows: Survivin: F: 5′-AGAACTGGCCCTTCTTGGAGG-3′, R: 5′-CTTTTTATGTTCCTCTATGGGGTC-3′ [13]; BAMBI: F: 5′-CGATGTTCTCTCTCCTCCCAG-3′, R: 5′-AATCAGCCCTCCAGCAATGG-3′ [24]. The sequences of GAPDH primers were: F: 5′-CGCATGGACTGTGGTCATGA-3′, R: 5′-TTCACCACCATGGAGAAGGC-3′ [25]. The relative expressions of cytokine genes were calculated using comparative Ct (2−ΔΔCT) analysis methods and assayed by the CFX96 QPCR machine (BioRad, USA), as in the following equations:
$$\begin{aligned} & {\text{Relative }}\;{\text{expression}} = \, 2^{ - \Delta \Delta C}_{T} \\ & \Delta \Delta Ct \, = \, \Delta Ct \, \left( {{\text{buccal }}\;{\text{sample}}} \right) \, {-}\Delta Ct \, \left( {{\text{PDL }}\;{\text{sample}}} \right) \\ & \Delta Ct = {\text{ AVG}}{. }Ct \, \left( {{\text{gene }}\;{\text{of }}\;{\text{interest}}} \right) \, {-}{\text{AVG}}{. }Ct\left( {{\text{housekeeping }}\;{\text{gene,}}\;{\text{ GAPDH}}} \right) \\ \end{aligned}$$
Data analysis
Data collection and results analysis were completed under blind code. The results are expressed as mean ± SEM. Statistical analysis was carried out with the SPSS 19 package (SPSS Inc., Chicago, USA). Differences between exposed groups were analyzed using independent Student’s t test, while multiple comparisons among more than two groups (as per addiction habits and call duration) were made by Tukey’s multiple comparison test.
