Pyrosequencing96 pyrosequencer

DNA was extracted from frozen tumor tissues or formalin-fixed paraffin-embedded (FFPE) tissues using a DNeasy Blood & Tissue Kit (Qiagen, Tokyo, Japan), and bisulfite modification of genomic DNA (500 ng) was performed using an EZ DNA methylation kit (Zymo Research, Orange, CA, USA). Pyrosequencing primers were designed to cover 16 CpG sites (CpG74–89) of the MGMT promoter gene [10 (link)]. Pyrosequencing of IDH1/2 and MGMT promoter genes was performed using PyroGold Q96 SQA Reagents and PyroMark Q96 software (version 2.5.7) on a pyrosequencing96 pyrosequencer (Qiagen, Tokyo, Japan) according to the manufacturer’s recommendations. The data were analyzed using PyroMark Q96 software, as described previously [10 (link), 11 (link)]. The mean percentage of MGMT promoter methylation was calculated by averaging 16 CpG islands (74–89) and was analyzed by the pyrosequencing method as described previously [10 (link)].

DNA was extracted from frozen tumor or formalin-fixed paraffin-embedded (FFPE) tissues using DNeasy Blood & Tissue Kits (Qiagen, Tokyo, Japan), and bisulfite modification of 500 ng genomic DNA was performed using EZ DNA Methylation Kits (Zymo Research, Orange, CA, USA). Pyrosequencing primers were designed to cover 16 CpG sites (CpG 74–89) of the MGMT promoter [10 (link)]. Pyrosequencing of IDH1/2 and MGMT promoter was performed using PyroGold Q96 SQA Reagents and PyroMark Q96 software (version 2.5.7) in a pyrosequencing96 pyrosequencer (Qiagen, Tokyo, Japan), according to the manufacturer’s instructions. Data were analyzed using PyroMark Q96 software, as described previously [10 (link),11 (link)]. MGMTpm% was calculated by averaging 16 CpG islands (74–89) and analyzed using the pyrosequencing method, as described previously, with some modifications [10 (link)].