Prior to conducting this experiment, the following minimal necessary sample was calculated using G-Power calculator: two groups of different temperatures, 6 measurements over time, an 80% power (beta = 0.20), a 95% level of significance (alpha 0,05) and an effect size of 0.5. The minimal sample size required to reject the null-hypothesis was six.
Upon the receipt of informed written consent from the six healthy participants, a pre-moistened sterile cotton swab (sterile Millipore water) was used to collect the participants’ palmar skin cells. Volunteers with skin diseases were excluded (Alessandrini et al., 2003). Six skin swabs were collected from each participant: 3 from each hand (Visser et al., 2011). The participants’ shedder statuses were nearly impossible to determine (Phipps and Petricevic, 2007). However, some known factors increased the amount of skin markers available for transfer (e.g. preventing hand washing prior to the collection and applying the cotton swab with pressure and friction) (Goray et al., 2010).
These swabs were divided into two groups; Group 1 was stored at 25 °C and Group 2 was stored at 40 °C. LCE1D mRNA expression levels were detected after 0, 1, 2, 3, 4 and 5 days. Measuring the LCE1D mRNA expression levels required the following three steps, according to Hanson et al. (2012):
RNA extraction from samples using SV Total RNA Isolation System (Z3100), promega, Madison, USA.
Intact RNA isolation requires four essential steps: the effective disruption of cells, the denaturation of nucleoprotein complexes, the inactivation of endogenous ribonuclease (RNase) activity and the removal of contaminating DNA and proteins. All these steps were performed according to the manufacturer’s protocol.
Conversion of RNA into cDNA using PCR Reverse Transcription System (A5001), promega, Madison, USA.
The extracted RNA was reverse transcribed into cDNA. Its yield and quality were determined at wavelength 260 nm using a UV2300 spectrophotometer (Indiamart, India).
Detection and quantification of the amplified genes using real time PCR Brilliant III SYBR® Green QRT-PCR Master Mix (600886), Stratagene Division, Agilent, Europe.
The amplified gene was determined via real time quantitative PCR (qPCR). The real time- PCR reaction mixture was 50 μl and consisted of 25 μl SYBR Green Mix (2×), 0.5 μl kidney cDNA, 2 μl primer pair mix (5 pmol/ μl each primer), and 22.5 μl H2O.
The used primers’ sequences were as follows (Bauer et al. 2003):
LCE1D Forward 5-CCTGTGCTGCCTGTGACT-3
Glyceraldehyde 3-phosphate dehydrogenase (GAPDH)
The PCR program set up was as follows: 1 cycle for 2 min. at 50 °C, 1 cycle for 10 min. at 95 °C, 1 cycle for 15 s at 95 °C, then 30 s at 60 °C followed by 30 s at 72 °C repeated 40 times and 1 cycle for 10 min. at 72 °C. The real time- PCR results were analyzed according to step one applied biosystem software. The normalized LCED (dCt) value of each sample was calculated as the difference between the Ct value of GAPDH (internal housekeeping gene) and LCE1D (Visser et al., 2011).
The data were coded and analyzed using the Statistical Package for Social Sciences(SPSS) version 21. The data were summarized using mean and standard deviation. Comparisons between values measured after 0, 1, 2, 3, 4 and 5 days were conducted using paired t-test and Repeated Measures ANOVA. P-values less than 0.05 were considered statistically significant.