Verity 96 well thermal cycler

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)].

Bacteria were cultured in LB broth overnight at 37°C, and 5–10 mL of culture were pelleted at 250 rpm. Plasmid DNA was purified with a QIAprep® Spin Miniprep Kit (Qiagen; H, Germany). Mobilization of DNA into E. coli K-12 was performed via electroporation (BMC Harvard Apparatus; MA, USA) at 1800 V. DNA amplification was carried out using PCR Master Mix (Promega; WI, USA) or Platinum® Taq DNA Polymerase High Fidelity (Invitrogen, California, USA) in a Verity 96-well thermal cycler (ThermoFisher Scientific; MA, USA). Purification of digested fragments was performed using a DNA Clean & Concentrator™ Kit (Zymo Research; CA, USA). DNA electrophoresis was carried out in 1% agarose gel with TAE 1x buffer, following by ethidium bromide staining and analysis using a BIORAD Chemi Doc® (Bio-Rad Laboratories; CA, USA).

The 1p/19q co-deletion status was determined by FISH as described by Snuderl et al. (29 (link)). Formalin-fixed paraffin-embedded tumor slides were prepared, and hematoxylin-eosin (HE) staining was performed to identify the tumor area. Two separate FISH were performed using SPEC Dual Color Probe (Zytovision, Germany), with chromosome 1p36 labeled orange and 1q25 labeled green on one slide, and chromosome 19p13 labeled green and 19q13 labeled orange on the other slide. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI), and fluorescent signals of 100 non-overlapping nuclei within the tumor area were measured using a BX41 TRF fluorescence microscope (Olympus, Japan). Glioma was defined as “1p (or 19q) loss” if the ratio of 1p: 1q (or 19q: 19p) was <0.75; otherwise, the tumor was recognized as “1p (or 19q) intact.”
IDH1 and IDH 2 mutations were detected by direct sequencing as described by Horbinski et al. (30 (link)). In brief, DNA was extracted from formalin-fixed paraffin-embedded tumor tissue using a Simlex OUP® FFPE DNA extraction kit (TIB, China), and subsequent PCR was performed using a Verity 96-Well Thermal Cycler (ThermoFisher, US) to amplify DNA fragments that contain IDH mutation hotspots (IDH1 R132 and IDH2 R172). The PCR products were then purified and sequenced using the Genetic Analyzer 3500 (ThermoFisher, US).