Methyl primer express

DNA was obtained using Nucleospin Tissue kit (Macherey-Nagel, 740952.5) as described in the user protocol. Bilsulfite treatment was carried out with 1 μg DNA, using Epitect Bisulfite Kit (Qiagen, 59104). CpG islands were detected and bisulfite sequencing primers for TAGLN gene were designed using the Methyl Primer Express® software (Applied Biosystems, USA). The primers amplified the region between −290 to +117 bp with respect to TSS (GenBank: NM_003186.3) (Left primer: 5′-GGGGTTAGAGAATAGTGAAGTAGGAGTA-3′; Right primer: 5′-ACACTCACAAAACTTCCTCAAAACT-3′). Gel extraction was carried out with the QIAquick Gel extraction kit (Qiagen, 28704). Specific PCR products were cloned into pGEM-T-easy cloning vector (Promega, A1360) after which plasmid isolation of selected colonies (5 for each sample) was performed with the PureLink Quick Plasmid Miniprep Kit (Invitrogen, K210011) and sent for sequencing. Sequencing of the bisulfite-treated DNA inserts was performed with SP6 primers using the dideoxy chain termination method (by Iontek, Istanbul, Turkey). Bisulfite sequencing results were analyzed using QUMA software [83 (link)]. Statistical differences between breast cancer and NTB cell lines were tested with Mann–Whitney test. Statistical differences between methylation percentages of paired tissues were tested with Wilcoxon signed rank test, in GraphPad Prism 6.0.

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′.