Cpgenome dna modification kit

The genomic DNA (1 μg) was bisulfite-modified using CpGenomeTM DNA Modification
Kit (S7820, CHEMICON, American) according to the manufacturer’s recommendations
and dissolved in 30 μL of nuclease-free water. The methylation and
non-methylation primers and their optimal annealing temperatures for
WT1, NKX6-1 and DBC1 are listed in Table 1. In vitromethylated DNA (IVD) was used as the positive control.

Total DNA was extracted using a Multisource Genomic DNA Miniprep Kit (Axygen) according to the manufacturer's protocol. A 1 μg amount of genomic DNA from each sample was modified with sodium bisulfite using the CpGenomeTM DNA Modification Kit (Chemicon). β-actin was used to normalize DNA inputs; a region of β-actin devoid of any CpG dinucleotide was amplified. The primer sequences were listed in Supplementary Table S1.

To determine if cordycepin affects tumor suppressor gene methylation in SNU719 cells, a methylation-specific PCR assay was performed using DNA subjected to bisulfite conversion. Following lysis and sonication by using a Bioruptor sonicator (5 min, 30 s on/off pulses), gDNA was extracted from SNU719 cells treated with 125 μM cordycepin. We used the CpGenome DNA Modification Kit (Millipore, Billerica, MA, USA) for sodium bisulphite conversion of the DNA. The sequences of the BCL7A primers used are shown in Table 1. Each 25-μl reaction contained 5 μl of bisulfite-treated DNA template, 5 μl of 5× reaction mix (NanoHelix), 5 μl of 5× TuneUp solution (NanoHelix), 1 μl of Taq-plus polymerase (NanoHelix), and 2.5 μl of 10 μM forward/reverse primer. Primers were specific for methylated and unmethylated BCL7A. The primer pairs specific for regions upstream and downstream of Cp and Wp (Cp/Wp) as well as upstream and downstream of Fp and Qp (Fp/Qp) promotors are listed in Table 1. The following cycle conditions were used: 95°C for 3 min; 30 cycles of 95°C for 30 s, 55°C for 30 s, and 72°C for 30 s; followed by 72°C for 10 min. The reactions were performed using a TaKaRa PCR Thermal Cycler and then run on a 1.5% agarose/TBE gel.

Promoter methylation status of the O(6)-methylguanine-DNA methyltransferase (MGMT) gene was analyzed in all cases using the CpGenome DNA Modification Kit followed by CpG-WIZ MGMT-Methylation specific PCR assay (Millipore Corporation, Billerica, MA, USA) following manufacturer instruction.

Genomic DNA of the OCSPCs and tumors was isolated with a Genomic DNA kit (Geneaid Biotech, Bade City, Taiwan). The DNA was converted with sodium bisulfite using a CpGenome DNA modification kit (Millipore, MA, USA), purified and amplified by PCR with ThermoHotStart 2X Gold PCR Master mix (Applied Biosystems) with primers (Additional file 2: Table S2) at 95 °C for 10 min, followed by 40 cycles of 95 °C for 30 s, 62 °C for 30 s, and 72 °C for 40 s, with a final extension at 72 °C for 10 min and holding at 4 °C. Bisulfite-modified, Sss I-treated normal lymphocyte DNA served as the positive methylated control, and bisulfite-treated normal lymphocyte DNA served as the unmethylated control.