Primer express software ver 3

Five genes (Edc4, Kiss1, Mrpl38, Stard3 and Zfp74) were selected for quantitative methylation-specific PCR analysis with the DNA samples isolated from the 0-ppm controls and the 1200-ppm IDPN-exposed group on PND 21. The isolated genomic DNA was sonicated using a Bioruptor UCD-250 sonicator (Cosmo Bio Co., Ltd.), mixed with incubation buffer, and then denatured with heat. Twenty percent of the mixture was stored as input DNA at -20°C until use. The remaining mixture was incubated with MBD2/magnetic bead complexes and then eluted. The methylation-enriched DNA was purified using an EpiXplore Methylated DNA Enrichment kit (Clontech Laboratories, Inc.). Input and methylation-enriched DNA samples were used as templates for quantitative measurement of methylation at target CpG sites by real-time PCR using Power SYBR ® Green PCR Master Mix (Thermo Fisher Scientific) and a StepOnePlus Real-time PCR System (Thermo Fisher Scientific). The PCR primers for the target gene CpG sites were designed using Methyl Primer Express software Ver. 1.0 (Thermo Fisher Scientific) and Primer Express software Ver. 3.0 (Supplementary Table 2; Thermo Fisher Scientific). The quantification was based on the comparative C T method and involved a comparison of the C T values of the methylation-enriched DNA to the C T values of the input DNA.