Sodium bisulfite

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	The methylation status of the target genes was determined using a methylation-specific PCR with primers specific for the methylated and unmethylated alleles of each gene after treating the genomic DNA with sodium bisulfite –. The primer sequences, annealing temperatures, and the expected sizes of PCR products are summarized in . Briefly, 1 µg of DNA was denatured with sodium hydroxide and modified with sodium bisulfite and DNA samples were purified using Wizard DNA purification resin (Promega). The sample DNA was treated with sodium hydroxide again, precipitated with ethanol, and resuspended in distilled water. All PCR amplification steps were carried out using reagents supplied in a GeneAmp DNA Amplification Kit with AmpliTaq Gold (PE Applied Biosystems) on PTC-100 (MJ Research).

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The methylation status of the target genes was determined using a methylation-specific PCR with primers specific for the methylated and unmethylated alleles of each gene after treating the genomic DNA with sodium bisulfite –. The primer sequences, annealing temperatures, and the expected sizes of PCR products are summarized in . Briefly, 1 µg of DNA was denatured with sodium hydroxide and modified with sodium bisulfite and DNA samples were purified using Wizard DNA purification resin (Promega). The sample DNA was treated with sodium hydroxide again, precipitated with ethanol, and resuspended in distilled water. All PCR amplification steps were carried out using reagents supplied in a GeneAmp DNA Amplification Kit with AmpliTaq Gold (PE Applied Biosystems) on PTC-100 (MJ Research).

Publication protocol

Methylation-specific PCR
A549 cells were transfected with plasmid constructs of Tnp cloned in the pcDNA™6.2/N-EmGFP-DEST vector and the empty destination vector. After 48 h of incubation, genomic DNA was purified using SolGent™ Genomic DNA prep kit (SolGent, Korea). The methylation status of the target genes was determined using a methylation-specific PCR with primers specific for the methylated and unmethylated alleles of each gene after treating the genomic DNA with sodium bisulfite –. The primer sequences, annealing temperatures, and the expected sizes of PCR products are summarized in . Briefly, 1 µg of DNA was denatured with sodium hydroxide and modified with sodium bisulfite and DNA samples were purified using Wizard DNA purification resin (Promega). The sample DNA was treated with sodium hydroxide again, precipitated with ethanol, and resuspended in distilled water. All PCR amplification steps were carried out using reagents supplied in a GeneAmp DNA Amplification Kit with AmpliTaq Gold (PE Applied Biosystems) on PTC-100 (MJ Research). The CpGenome™ universal methylated and unmethylated DNA was used as a positive control for the methylated and unmethylated genes, respectively. PCR products were analyzed on 2% agarose gel and stained with ethidium bromide. Each MSP was repeated at least once to confirm the results.

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