Patients and tumour samples
Specimens excised from 80 patients with high-grade glioma (69 cases of glioblastoma and 11 cases of anaplastic astrocytoma) at Kyoto University Hospital or nearby regional hospitals between 1998 and 2008 were stored at -70°C until use. All histological diagnoses were performed in the Kyoto University Pathology Unit according to the 2000 or 2007 WHO classifications.
Sixty of the 80 samples were recruited from those used in the previous study . They were collected from patients enrolled in a phase II clinical trial using nimustine, carboplatin, vincristine, and IFN-β with radiotherapy for high-grade gliomas (the KNOG study) . The remaining 20 patients were treated with temozolomide and radiotherapy. The learning set included 44 samples (43 glioblastoma, 1 anaplastic astrocytoma) from the KNOG study. Recurrence was detected in 36 of the 44 patients and their median progression-free survival was 7 months. The test set included 36 samples (26 glioblastoma and 10 astrocytoma). Twenty-three of the 36 patients showed tumour progression, and their median progression-free survival was 8 months.
Institutional approval for this study was obtained from the Institutional Review Board of Kyoto University, and informed consent was obtained from all patients prior to surgery.
RNA extraction and cDNA synthesis
Total RNA was isolated from 100 mg of the tumour specimen using TRIzol (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. RNA concentrations and A260/A280 ratios were measured using a NanoDrop ND-1000 (NanoDrop Technologies, Montchanin, DE, USA). Only RNA samples with A260/A280 ratios above 1.90 were included in the study. RNA integrity was confirmed by analysis with the Agilent 2100 bioanalyser.
After DNase treatment, 5 μg of total RNA in 10 μl of distilled water was incubated with 1 μl of oligo(dT) primer for 5 min at 70°C. Total RNA was reverse transcribed in a total volume of 20 μl containing 4 μl of 5× first strand buffer, 1 μl of RNase inhibitor (Invitrogen), 2 μl of 0.1 M DTT, 0.5 μl of 20 mM dNTP and 1 μl of SuperScript III Reverse Transcriptase (Invitrogen). The samples were incubated at 45°C for 1 hr. Next, a reaction mixture (total volume of 103 μl) containing 10 μl of 10× Escherichia coli (E. coli) ligation buffer, 2 μl of 20 mM dNTPs, 2 μl of 0.1 M DTT, 2 μl of E. coli ligase (Invitrogen), 1 μl of RNase H (Invitrogen), 4 μl of E. coli DNA polymerase (Invitrogen) and 82 μl of nuclease-free water was added. The resulting reaction mixture was incubated at 16°C for 120 min and then at 70°C for 20 min. The reaction mixture was then diluted five-fold with nuclease-free water and stored at -30°C until RT-PCR analysis.
Quantitative real-time reverse transcription-PCR
Quantitative PCR amplification assays were performed by a SYBR Green fluorescent assay using the ABI PRISM 7500 real-time PCR sequence detection system (Applied Biosystems, Foster City, CA, USA). Reactions were performed in a 96-well plate with 20-μl reaction solutions containing SYBR Premix Ex Taq II (10 μl) (Takara Bio., Inc., Japan), ROX reference dye II (0.4 μl), 10 μM forward and reverse primers (0.8 μl), 1 μl of cDNA template, and nuclease-free water (7 μl). Cycling conditions included an initial denaturation for 10 sec at 95°C, followed by 40 cycles of 5 sec at 95°C and 34 sec at 60°C. For determination of the reference gene, a standard curve was generated for each assay using seven serial dilutions of an amplified human brain cDNA library ranging from 20 ng to 20 fg.
The delta-delta Ct method was employed for the diagnostic assays. Ct values were calculated following the manufacturer's instructions (Applied Biosystems, Foster City, CA, USA), using UBL5 as the internal reference. The diagnostic genes fulfilled the criterion that the absolute value of the slope of the log input amount vs. Δ
Ct should be < 0.1.
Thirty primers for the selected gene candidates and for the internal and negative controls were added in triplicate to 96-well plates, and the samples were measured using one plate per sample. The negative controls showed no detectable amplification or background levels of amplification (Ct ≥ 37, compared with 16 to 31 with sample DNAs). The mean and the standard deviation of differences of Ct values between duplicates were 0.060 and 0.086, respectively. Sequence detection software (Applied Biosystems) results were exported as tab-delimited text files and imported into Microsoft Excel for further analysis.
Statistical data processing was performed using Excel and SPSS, and Pearson's correlation coefficients (r) were computed for each cross-platform comparison. Progression-free survival was measured from the day of surgery to the time of the first event of progression or to the last day of follow-up, according to the Kaplan-Meier method. Curves were compared using the log-rank test.