Seqqunom's (San Diego, CA, USA) MassARRAY platform was used to perform the quantitative methylation analysis. This system utilizes MALDI-TOF mass spectrometry in combination with RNA base-specific cleavage (MassCLEAVE kit). A detectable pattern is then analyzed to determine the methylation status. PCR primers were designed using EpiDesigner (Sequenom). Amplicons were designed to cover the CpG sites from an approximately 4500-bp region upstream of the GFAP transcription start site to the intron 1 region. For each reverse primer, an additional T7 promoter tag for in vivo transcription was added, as well as a 10mer tag on the forward primer to adjust for differences in melting temperatures. The PCRs were carried out in a 5-ml format with 10 ng/ml bisulfite-treated DNA, 0.2 units of HotStart Taq DNA polymerase (Qiagen), 16 supplied HotStart buffer, and 200 mM PCR primers. Amplification of the PCR was as follows: preactivation at 95uC for 15 min, 45 cycles of 95uC denaturation for 20 s, 56uC annealing for 30 s, and 72uC extension for 30 s, with a final 72uC incubation for 4 min. Dephosphorylation of unincorporated dNTPs was performed by adding 1.7 ml of H 2 O and 0.3 units of shrimp alkaline phosphatase (Sequenom), followed by incubation at 37uC for 20 min and then at 85uC for 10 min to deactivate the enzyme. Next, in vivo transcription and RNA cleavage were achieved by adding 2 ml of the PCR product to 5 ml of the transcription/cleavage reaction and incubation at 37uC for 3 h. The transcription/cleavage reaction contained 27 units of T7 R&DNA polymerase (EpiCentre, Palmerston North, New Zealand), 0.646 of T7 R&DNA polymerase buffer, 0.22 ml T Cleavage Mix (Sequenom), 3.14 mM DTT, 3.21 ml H 2 O, and 0.09 mg/ml RNaseA (Sequenom). Reactions were additionally diluted with 27 ml of H 2 O and conditioned with 6 mg of CLEAN Resin (Sequenom) for optimal mass-spectrum analysis. Samples were then dispensed with the MassARRAY nanodispenser (Samsung, Irvine, CA, USA) on a 384-well SpectroChip (Sequenom). Mass spectra were acquired using a MassARRAY Compact MALDI-TOF (Sequenom), and the methylation ratios were calculated by comparing the difference in spectra intensity derived from methylated and non-methylated template DNA by the Epityper software version 1.0 (Sequenom).