Page 99 - Molecular features of low-grade developmental brain tumours

THE CODING AND NON-CODING TRANSCRIPTIONAL LANDSCAPE OF SEGA
transcription kit (Applied Biosystems, Foster City, CA, USA) according to the manufacturer’s instructions, and the PCRs were run on a Roche Lightcycler 480 thermocycler (Roche Applied Science, Basel, Switzerland).
Quantification was performed using the LinRegPCR software in which linear regression on the Log (fluorescence) per cycle number data is applied to determine the amplification efficiency per sample 63,64. The starting concentration (predicted by the LinRegPCR prediction model) of each specific mRNA product was divided by the starting concentration of the reference gene Elongation factor 1-alpha 1 (EEF1A1) and this ratio was compared between groups. The starting concentrations of miRNA-20a-5p, miRNA-34a-5p, miRNA-130b-3p and miRNA-181a-5p were divided by the geometric mean of the starting concentrations of reference genes [the U6B small nuclear RNA gene (RNU6-6P) and RNU44 (SNORD44)] and this ratio was compared between groups.
Immunohistochemistry
Tissue from control brain and SEGA was fixed in 10% buffered formalin and embedded in paraffin. Paraffin-embedded tissue was sectioned at 6 μm, mounted on pre-coated slides (Star Frost, Waldemar Knittel GmbH, Brunschweig, Germany) and stained with hematoxylin and eosin (HE) for the morphological evaluation. Sections of the most representative paraffin-embedded specimen of each case were used for double labeling. Sections were deparaffinated in xylene, rinsed in ethanol (100%, 95% and 70%) and incubated for 20 minutes in 0.3% hydrogen peroxide (H2O2) in methanol to block endogenous peroxidase activity. Antigen retrieval was performed by incubating the sections in 0.1 M citrate buffer pH 6.0 at 120°C for 10 min using a pressure cooker. Sections were washed with phosphate buffered saline (PBS, pH 7.4) and incubated with 10% normal goat serum (NGS) for 30 minutes at room temperature. Primary antibodies (1:200 rabbit monoclonal IgG anti- LAMTOR1, 8975, Cell signaling, Danvers, MA) in Normal Antibody Diluent (Immunological, Duiven, The Netherlands) were incubated overnight at 4°C. After washing with PBS, sections were incubated with Brightvision poly-alkaline phosphatase (AP)-anti-rabbit (Immunologic, Duiven, The Netherlands) for 30 minutes at room temperature. AP activity was visualized with the AP substrate kit III Vector Blue (SK-5300, Vector laboratories Inc., Burlingame, CA, USA). To remove the first primary antibody sections were incubated at 120°C in citrate buffer (10 mM NaCi, pH 6.0) for 10 min. Incubation with the second primary antibody (1:400 rabbit monoclonal IgG anti-phospho-ERK1/ERK2, 4370S, Cell signaling, Danvers, MA; 1:200 rabbit monoclonal IgG anti-pS6, 4857, Cell signaling, Danvers, MA) was performed overnight at 4°C. Sections were stained with a polymer based peroxidase immunohistochemistry detection kit (Brightvision plus kit, ImmunoLogic, Duiven, The Netherlands) according to the manufacturer’s instructions. Staining was performed using 3-amino-9-ethyl carbazole (AEC, Sigma-Aldrich, Munich, Germany) in 0.05 M saline buffer pH 4.9 with 0.01% H2O2.
Astrocyte and SEGA cell cultures
Primary fetal astrocyte-enriched cell cultures were obtained from human fetal brain tissue
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