Blood & Cell Culture DNA Midi Kit (25)

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Methylation Analysis by Bisulfite Genomic Sequencing: Genomic DNA was isolated from cells using the Blood and Cell Culture DNA Mini Kit (Qiagen). The bisulfite reaction was performed using the CpG Genome Modification Kit according to the manufacturer's protocol (Intergen). In each bisulfite reaction, 1 μg of DNA was used initially. The DNA was ultimately eluted in 30 μl of TE. Each PCR contained 5 μl of bisulfite-modified DNA in a final volume of 50 μl of PCR mixture composed of 5 μl of 10× PCR Buffer (Invitrogen), 1.5 μl of 50 mm MgCl2(Invitrogen), 1 μl of 10 mm of each dNTP (peqLAB), 1 μl of each 100-pmol primer, and 4 units of DNA Taq polymerase (Invitrogen). The first set of primers was designed after taking into account the bisulfite conversion reaction. Primer sequences were as follows: cav1prom1s (−877 → −848) 5′-tgtgtattttgtaaatatggtataatttg-3′ (sense) and cav1prom1as (−547 → −525) 5′-CCATCTCTACCTTAAAACACAT-3′ (antisense). The second pair of primer was cav1prom3s (−477 → −455) 5′-GGATAGGGTAGGATTGTGGATT-3′ (sense) and cav1prom3as (−223 → −202) 5′-CACATCCCCAAAATTCTAACA-3′ (antisense) (36). The nucleotide positions are numbered relative to translation start codon (+1). PCR was performed at 95 °C for 5 min, 35 cycles at 95 °C for 30 s, 58 °C for 40 s, and 72 °C for 50 s and a final extension at 72 °C for 5 min. The amplified fragments were gel-purified and subcloned into the pGEM T-Easy vector (Promega). Plasmids were purified using the QIAprep Spin MiniPrep Kit (Qiagen), and individual clones were sequenced by TopLab (Martinsried, Germany). In addition, amplified fragments derived from primer set cav1prom3s/3as were directly sequenced with use of the corresponding primers. After modification, unmethylated cytosines appear as thymidines, whereas methylated cytosines remain unchanged.

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