Koos Boeve

150 Chapter 8 DNA was extracted from sections and saliva cell pellets by phenol-chloroform extraction and ethanol precipitation as described previously [29]. Samples were dissolved in TE-4 buffer (50 µl for FFPE and FF, 300 µl for saliva) and stored at 4°C. DNA quality and quantity was assessed using the Nanodrop and Biomed II PCR protocol [30]. Bisulfite treatment and Quantitative Methylation Specific PCR (qMSP) Isolated DNA was treated with bisulfite for methylation-specific-PCR (MSP) as previously described [23,29]. Briefly, bisulfite treated DNA (bisDNA) was acquired using the EZ DNA methylation kit (Zymogen, BaseClear, Leiden, The Netherlands), according to the manufacturer’s protocol. Methylation-specific-PCR (MSP) was performed on 20 ng bisDNA as follows: 10 min 95°C, 40 cycli (1 min 95°C, 1 min T annealing , 1 min 72°C), followed 10 min 72°C and ∞ 4°C. Primer sequences and T annealing are summarized in Table 2. As controls in each qMSP, leukocyte DNA from healthy individuals (as a control for endogenous methylation), leukocyte DNA that was in vitro methylated (I.V.) by SssI methyltransferase (New England BioLabs Inc., Bioké, Leiden, The Netherlands) (as a control for methylated DNA) and leukocyte DNA that was amplified according to manufacturer’s protocol using whole genome amplification with the Illustra Ready-To-Go GenomiPhi HY DNA Amplification Kit (GE Healthcare, Little Chalfont, UK) (as a control for hypomethylation). Cytosine conversion by bisulfite treatment was checked with primers specific for bisulfite treated Beta-Actin (ACTB) and DAPK as described earlier [31,32]. After MSP, PCR products were separated and visualized by custom Ethidium Bromide staining. QMSP was performed as previously described with an internal dual-labeled hybridization probe (IDT, Coralville, IA) [29,31]. For CMTM2 and FERMT3 no specific primers and probes within 250 bp of the methyl core region could be designed. For four genes ( C11orf85 , KCNA5 , SIPA1 and TBX4 ), QMSP primers and probes were designed by Methyl Primer Express TM Software v1.0 (Thermo Fisher Scientific, Applied Biosystems, Leiden, The Netherlands) and checked using Clone Manager software (Sci-Ed software, Denver, USA) (Table 2). Serial dilutions of I.V. DNA were used to calculate standard curves for each primer-probe set, resulting in suitable conditions for the detection of methylation of C11orf85, KCNA5 and SIPA1 . For TBX4 no optimal condition was found and therefore TBX4 was excluded for further analysis. The amount of bisulfite treated DNA input of each sample was determined by qMSP for ACTB (Table 2) as reported previously [31]. Fluorescence was measured in triplicates for 50 cycles using the following mixture: 7.5 µl of 2* LightCycler 480 Probes Master mix (Roche Diagnostics GmbH, Mannheim), 300 nM of forward and reverse primers (IDT, Coralville, IA), 200 nM of probe (IDT) and 2.5 µl bisulfite-modified DNA (~25 ng). Each sample was analyzed by LightCycler 480 (Roche Diagnostics GmbH, Mannheim). Relative methylation