Patient and control participants
This experimental study was conducted in the genetics Laboratory of Tarbiat Modares University (Tehran, Iran) within 2013-2014. In this research, 61 MS patients and 36 age-, race-, and sex-matched controls were recruited from Multiple Sclerosis centers at Sina Hospital (Tehran, Iran) to investigate the mRNA expression level of TGF-β1, TGF-β2, TGF-β-R1 and TGF-β-R2 genes. The patients were diagnosed according to the McDonald criteria. informed consent was obtained from all human adult participants and the study was approved by the Ethics Committee of Tarbiat Modares University (ethical code: d52/6723).
RNA extraction and cDNA synthesis
After blood collection, PBMC separation was performed using density gradient Ficoll/Paque solution (lympholyte, Cedarlane, Netherlands) and total RNA was extracted from all patient and control samples, using RNXTM-plus reagent (Cinnagen, Iran) according to the manufacturer’s protocol. The samples were subsequently reverse-transcribed into cDNA, using 3 µg total RNA and 250 μg oligo dT (MWG, Germany). The reaction was incubated at 70° C for 10 minutes and cooled on ice for 3-5 minutes, followed by adding RNase inhibitor, 10 mM dNTPs and Reverse Transcriptase (all from Fermentas, Canada) to 20 ml total volume of reaction mixture. The mixture was ultimately incubated at 42° C and 80° C for 60-90 and 15 minutes, respectively.
Quantitative reverse transcriptase PCR
cDNA for each sample was used to evaluate the mRNA expression level of TGF-β1, TGF-β2, TGF-β-R1 and TGF-β-R2 genes using relative quantitative reverse transcriptase PCR (qRT-PCR). In this experiment, glyceraldehyde3-phosphate dehydrogenase (GAPDH) was utilized as housekeeping gene.
Each qRT-PCR reaction was performed in a final volume of 20 µl, using 10 ng cDNA, 2x SYBR Green I master mix (Takara, Shiga, Japan) and appropriate primer pair set (Table S1). Thermal condition, as one-step RT-PCR, was carried out by an initial step at 95° C for 15 minutes, followed by 40 cycles at 95° C, 60° C and 72° C for 15 seconds, 30 seconds and 30 seconds, respectively, in Applied Biosystems 7500 Real-Time PCR System (Applied Biosystems, USA). Termed cycle threshold (Ct) was determined for each sample, and the average Ct of duplicate samples was calculated.
Statistical analysis
Relative quantification data analysis was performed using arbitrary method (ΔCt). The significance of differences between control and test groups was determined by independent t-test using SPSS software (Version 20; SPSS Inc, Chicago, USA) and GraphPad Prism 5 (GraphPad Software, Inc., San Diego, USA). The correlation analysis was assessed by Pearson’s correlation coefficient. A P-value of 0.05 was set as significant threshold.