Drr007 kit

The methylation status of the MGMT promoter was measured by pyrosequencing, as previously described [25 (link)]. Briefly, DNA was extracted from formalin-fixed, paraffin-embedded tumor samples with a Simplex OUP® FFPE DNA Extraction Kit (TIB, China) and quantified by spectrophotometry with a NanoDrop 2000 system (Thermo Fisher, US). Bisulfate modification was performed with an EpiTect Bisulfite Kit (Qiagen, Germany), and PCR was carried out with a DRR007 Kit (Takara, Japan) using a Verity 96-Well Thermal Cycler (Thermo Fisher, US). Pyrosequencing was subsequently performed in 10 CpG island regions within the MGMT promoter using the PyroMark Q96 system (Qiagen, Germany). Gliomas were defined as having a methylated MGMT promoter if the average methylation rate of the CpG regions was greater than or equal to 8%; gliomas were defined as having an unmethylated MGMT promoter if the average methylation rate was less than 8% [25 (link)].

IDH1 and IDH2 mutations were detected postoperatively in patient tumor tissue using direct sequencing, as described by Horbinski et al. (29 (link)). DNA was isolated from formalin-fixed, paraffin-embedded tumor tissue using the Simplex OUP®FFPE DNA extraction kit (TIB, China), and the quantity was assessed by spectrophotometry using a NanoDrop 2000 (Thermo Fisher, US). Polymerase chain reaction (PCR) was accomplished with IDH1 primer (IDH1-F) 5′-TGATGAGAAGAGGGTTGAG-3′, (IDH1-R) 5′-TTACTTGATCCCCATAAGCC-3′, and IDH2 primer (IDH2-F) 5′-GACCCCCGTCTGGCTGTG-3′, (IDH2-R) 5′-CAAGAGGATGGCTAGGCGAG-3′ using the DRR007 kit (Takara, Japan) and a Verity 96-Well Thermal Cycler (Thermo Fisher, US) to amplify the fragment that contains two mutation hotspots. PCR products were treated with Exonuclease I and Antarctic Phosphatase (New England Biolabs, UK) and sequenced using a Genetic Analyzers 3500 (Thermo Fisher, US).