Quantitative NEFH Methylation Assay

Quantitation of NEFH methylation was carried out by a quantitative real-time fluorimetric 5′ exonuclease PCR assay. The qMSP primers 5′-ACCCGACCGCGACGACTATA-3′ (forward), 5′-CGTCGAAGTTTATTATGGTTTGAGTAGG-3′ (reverse) and the Taqman® probe 5′-FAM-CGCCCTAATACTACCGCAATACCTCCCGC-BHQ-3 were created by use of the Beacon Designer™ software (PREMIER Biosoft, Palo Alto CA, USA). The qMSP analysis covered nine CpG sites on chromosome 22 at positions 29,876,165, ∼169, ∼171, ∼174, ∼206, ∼218, ∼231, ∼263, and ∼266 according to the GRCh37/hg19 annotation in the UCSC genome browser 31 (link),32 (link). Real-time PCRs were performed as duplicates on an ABI 7900HT (Life technologies, Foster City, CA, USA) in 384-well plates using an automated liquid handling system as described previously 20 (link). The experimenters carried out measurements without knowledge of type, order, clinicopathological or survival status of samples. Relative methylation levels were calculated as an analogue of the delta–delta C_t method by normalizing the difference of NEFH methylation-specific real-time detection and methylation independent internal QC1 control measurement with the corresponding difference for the fully methylated DNA control samples as described previously 20 (link).