Hin 6 1

Total DNA (600 ng) from fresh frozen tumour tissue (n=22) and PBMCs (n=8, 4 male and 4 female) was subjected to methylation-specific restriction enzymes (MSRE) to cleave unmethylated DNA. Methylated DNA remains uncleaved and can be subjected to screening using targeted DNA microarrays for 360 methylation marker candidates (targeting CpG islands and human gene promoters). The main principle of the methodology was published previously (Pulverer et al, 2012 (link)). For the experiments of the present study we used the MSREs HpaII (cut site: CCGG; Fermentas, St. Leon-Rot, Germany), Hin6I (cut site: GCGC; Fermentas, St. Leon-Rot, Germany), AciI (cut site: CCGC; NEB, Frankfurt am Main, Germany) and HpyCH4IV (cut site: ACGT; NEB, Frankfurt am Main, Germany). A unit of 3 U (0.3 μl) of each enzyme were used per digestion reaction and incubated at 37 °C for 16 h. The digested DNA samples were amplified in 16 multiplex PCR reactions amplifying in total 360 methylation marker candidates using biotinylated reverse primers. Pooled amplicons were detected on a targeted DNA microarray via streptavidin–Cy3 conjugate. Subsequently, significant markers were identified applying statistical tests for class comparison and class prediction. Primer sequences are available on request (Weinhäusel et al, 2009 ; Weinhäusel and Pulverer, 2013 ).