text: Isolation of mRNA and real-time PCR
  Total cellular RNA was extracted from PB CD4+ T cells using an RNA isolation kit (RNeasy Mini kit; Qiagen, Valencia, CA, USA), according to the manufacturer's instructions. cDNA was synthesized from total RNA with Molony murine leukemia virus reverse transcriptase (US Biochemical, Cleveland, OH, USA) and oligo-(dT)15 primers (Promega, Madison, WI, USA). Real-time PCR was performed with the LightCycler Instrument (Roche Diagnostics, Penzberg, Germany) in glass capillaries. The reaction mix containing Taq DNA polymerase and DNA double-strand-specific SYBR Green I dye (Lightcycler FastStart DNA Master SYBR Green I; Roche Diagnostics) and specific primers were added to cDNA dilutions. 
  The cDNA samples were denatured at 95degrees C for 10 min, and were then amplified for 40-50 cycles: at 95degrees C (10 s), at 65degrees C (15 s), and 72degrees C (22 s) for beta-actin; at 95degrees C (10 s), at 62degrees C (15 s), and at 72degrees C (10 s) for SOCS1; and at 96degrees C (10 s), at 68degrees C (15 s), and at 72degrees C (15 s) for SOCS3. Amplification curves of the fluorescence values versus cycle number were obtained, and a melting curve analysis was then performed. The levels of SOCS1 and SOCS3 expression were determined by normalizing relative to beta-actin expression. The forward and reverse primers were as follows: for beta-actin, 5'-GTGGGGCGCCCCAGGCACCA-3' and 5'-CTCCTTAATGTCACGCACGATTTC-3' ; for SOCS1, 5'-AGACCCCTTCTCACCTCTTG-3' and 5'-GCACAGCAGAAAAATAAAGC-3' ; and for SOCS3, 5'-CCCGCCGGCACCTTTCTG-3' and 5'-AGGGGCCGGCTCAACACC-3'. 
  