Ethical permit to perform this study was obtained, in accordance with the Helsinki Declaration, from the Ethics Committee of the Department of Surgery of the Hospital District of Helsinki and Uusimaa, Finland (decision #127/2005 issued on November 23, 2005). Each volunteer was informed in detail about all experimental procedures and each of them has signed the informed consent form (in Finnish language).
Exposure of volunteers to mobile phone radiation
Volunteers were exposed to 900 MHz GSM mobile phone radiation in an experimental setup described in detail elsewhere . The source of irradiation was a half-wave dipole fed with a computer controlled GSM phone. The specific absorption rate (SAR) induced in the skin was 1.3 W/kg what is below the ICNIRP safety guidelines (2.0 W/kg). During the exposure small area of the right forearms was irradiated for one hour. The other, non-irradiated forearm was used as sham control. Immediately after exposure skin punch-biopsies were taken from the exposed and non-exposed skin for protein analysis.
Protein extraction from skin biopsies
Skin punch biopsies, consisting of both dermis and epidermis but without the underlying fat tissue, were frozen immediately after harvesting in liquid nitrogen and stored at -80°C. Isolation and separation of proteins were performed in the blinded manner. Proteins were isolated from frozen skin using TRIzol® reagent protocol as described by the manufacturer (Invitrogen, Carlsbad, CA, USA) with a few modifications. Briefly, the chopped skin punch-biopsies were immersed in 0.5 ml of ice-cold TRIzol reagent and homogenized on ice with 70 strokes of the pestle in DUALL 1 ml tissue grinder (Kimble Chase Life Science and Research Products, Vineland, NJ, USA). After the phase separation of TRIzol reagent, the organic phase containing DNA and proteins was collected. DNA was then precipitated with ethanol and proteins were isolated from the phenol-ethanol supernatant. The proteins were then precipitated by isopropyl alcohol and pelleted at 12000 × g for 10 min at +4°C. The protein pellet was washed 3 times with 0.3 M guanidine hydrochloride solution in 95% ethanol and once with 99.5% ethanol. During the extraction pellets were grinded with pellet pestle in order to improve the solubility of the proteins. After each wash step, proteins were centrifuged 7500 × g for 5 min at +4°C. The air-dried protein pellet was dissolved in 2-DE rehydration buffer containing 9 M urea, 2% (w/v) CHAPS, 0.5% (v/v) IPG buffer pH 4–7 and 5 mg/ml DTT (added as fresh). The protein concentration of sample was measured using the Bradford method. The samples were stored at -80°C.
Protein separation with 2-DE
Proteins were separated by standard 2-DE. Briefly, the first dimension was performed in IPGphor™ (GE Healthcare, UK) isoelectric focusing (IEF) apparatus. Linear, 24 cm long, pH 4–7 Immobiline™ DryStrip gels (IPG-strips, GE Healthcare, UK) were rehydrated in the strip holders for 4 hours in 0.45 ml rehydration buffer containing 9 M urea, 2% (w/v) CHAPS, 0.5% (v/v) IPG-buffer pH 4–7, 1.2% (v/v) DeStreak™ reagent, a trace of bromophenol blue and 150 μg of total amount of protein. IEF was carried out at +20°C using following step-and-hold settings: 50 V, 8 h; 100 V, 1 h; 500 V, 1 h; 1000 V, 1 h; 2000 V, 1 h; 8000 V, until 95000 Vh was achieved. Then, the IPG-strips were incubated at room temperature in equilibration buffer (50 mM Tris-HCl, pH 8.8, 6 M urea, 30% (v/v) glycerol, 2% (w/v) SDS, a trace of bromophenol blue, and 10 mg/ml DTT) for 15 min and for another 15 min in the same buffer that contained 25 mg/ml of iodoacetamide instead of DTT. The second-dimension separation was performed using 9%SDS-PAGE gels. Electrophoresis was carried out at +10°C using an Ettan™ DALTsix electrophoresis unit (GE Healtcare) at a constant power of 3.5 W/gel for 0.5 h and then 13 W/gel until the dye front reached the bottom of the gel (about 4 h). The ready gels were silver stained to visualize protein spots. Stained gels were scanned into computer using GS-710 Calibrated Imaging Densitometer (Bio-Rad Laboratories, Hercules, CA, USA). The gels were analyzed using PDQuest 7.2 software (Bio-Rad).