SOCS3 Methylation-Specific PCR Protocol

Methylation-specific PCR (MSP) was performed based on results of a previous study (9 (link)). The methylation-specific primer was designed using the Methyl Primer Express software v1.0 (Applied Biosystems, Foster City, CA, USA). Primer sequences are shown in Table I. The PCR reaction conditions were as follows: Initial denaturation at 95°C for 10 min; 40 cycles of denaturation at 95°C for 40 sec; annealing at 65°C for 30 sec and elongation at 75°C for 40 sec. Finally, cycling was completed with an elongation step at 75°C for 10 min. Sequences of MSP primers in exon 1 of SOCS3 were: Sense, 5′-TTCGAGGTGTTCGATTAGAC-3′ and antisense, 5′-AAAATGCTTCCGACATAGAT-3′. Sequences of MSP primers in intron 1 of SOCS3 were: Sense, 5′-GCCTCCGGGTAAGCGTGGATTAG-3′ and antisense, 5′-AATACATAGAGGCTGCGCGAAGC-3′.