Page 94 - Molecular features of low-grade developmental brain tumours

4
92
CHAPTER 4
Materials and Methods
SEGA tumour specimens
A total of twenty-one SEGA specimens were obtained from the following sites: the Amsterdam UMC (location AMC), the University Medical Center Utrecht, University Medical Center Groningen, Medical University of Vienna, Children’s Memorial Health Institute in Warsaw and Meyer Children’s Hospital in Florence. Nineteen of the twenty-one SEGA samples included in this study were obtained from patients that met the clinical diagnostic criteria for TSC. From the twenty-one SEGA samples nineteen were selected and used for RNA-sequencing (RNA-Seq)(Table 1). When DNA material permitted, TSC1/TSC2 mutation analysis was performed as part of routine clinical care on blood or tumour sample DNA or was determined using massively parallel sequencing (including analysis of LOH) as described previously by Bongaarts et al., 2017 (Table 1) 24,44. Histological diagnosis was confirmed following the current WHO classification guidelines by two independent neuropathologists 8. The following clinical data was extracted from medical records: TSC1/TSC2 mutation status, gender, localization of the resected area, age at seizure onset, duration of active epilepsy, drug management at time of surgery (including treatment with mTORC1 inhibitors), size of the tumour, tumour recurrence/regrowth and presence of other TSC related malformations. No peri-tumoural tissue was available, therefore periventricular brain tissue was obtained (as well as one sample of cortex tissue) from autopsy controls without a history of TSC, epilepsy or brain tumours. A total of thirteen controls were obtained of which eight were selected for RNA-Seq and five were used for additional immunohistochemistry. Additionally, 4 cortical tubers, 1 angiomyolipoma (AML) and 1 sample of normal renal tissue were obtained from TSC patients who met the clinical diagnostic criteria for TSC (Supplementary Table 1). Specimens were obtained and used in accordance with the Declaration of Helsinki and this study was approved by the Medical Ethics Committees of each institution.
RNA isolation and RNA-Sequencing
For RNA isolation frozen tissue or cultured cells were homogenized with Qiazol Lysis Reagent (Qiagen Benelux, Venlo, The Netherlands). The total RNA including the microRNA (miRNA) fraction was isolated using the miRNeasy Mini kit (Qiagen Benelux, Venlo, The Netherlands) according to manufacturer’s instructions. The concentration of RNA was determined using a Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, USA) for cell cultures or Qubit® 2.0 Fluorometer (Life Technologies, Carlsbad, CA, USA) for frozen tissue. For RNA-Seq the RNA integrity was assessed using a Bioanalyser 2100 (Agilent). Library preparation and sequencing were completed at GenomeScan (Leiden, The Netherlands). The Illumina (San Diego, California, USA) RNA-Seq and TruSeq Small RNA-Seq sample preparation kits were used to prepare sequencing libraries of mRNA and small RNA in accordance to manufacturers guidelines. Clustering and DNA sequencing was performed using the Illumina cBot and HiSeq 2500 according to manufacturer’s protocols. Each library was subjected to paired-end sequencing, producing reads of 125 nucleotides in length with a read-depth of 36 million reads for RNA-Seq and 12 million reads for small RNA-Seq.