Page 157 - Molecular features of low-grade developmental brain tumours

MIR-519D & MIR-4758 CAN IDENTIFY GG FROM DNT AND ASTROCYTOMAS
Tissue preparation
Brain tissue from control and tumour patients was fixed in 10% buffered formalin and embedded in paraffin. Paraffin-embedded tissue was sectioned at 5 μm and mounted on pre-coated glass slides (Star Frost, Waldemar Knittel GmbH, Brunschweig, Germany). Sections of all specimens were processed for hematoxylin and eosin (HE staining), as well as for immunohistochemical stainings for a number of neuronal and glial markers to confirm the diagnosis. Additional tissue from controls (n=8) and from patients with GG (n=15), PA (n=15), AII (n=10), AIII (n=14) and GB (n=15) was snap frozen in liquid nitrogen and stored at -80°C until further use (RNA isolation for microRNA array and/or quantitive real-time PCR).
In situ hybridization
ISH for miR-519d and miR-4758 was performed using 5′ - 3′ double digoxygenin (DIG)-labeled Superior probes (miR-519d-3p; DIG-CacTcuAaaGggAggCacTuuG-DIG; miR-4758-3p: DIG- GagGguGguCagCagGugGggCa-DIG; Ribotask ApS, Odense, Denmark). The hybridizations were done on 5 μm sections of paraffin-embedded materials as described previously 30. The probes were hybridized at 58°C for 1 h, and the hybridization was detected with alkaline phosphatase (AP)-labeled anti-DIG (Roche Applied Science, Basel, Switzerland). NBT (nitro- blue tetrazolium chloride)/BCIP (5-bromo-4-chloro-3′-indolyphosphate p-toluidine salt) was used as chromogenic substrate for AP. Negative control assays were performed without probes and without primary antibody (sections were blank). For double-staining, combining immunohistochemistry with ISH, the sections were first processed for ISH and then processed for immunohistochemistry with glial fibrillary acidic protein (GFAP; monoclonal mouse, Sigma, St. Louis, Mo, USA; 1:4000), and NeuN (neuronal nuclear protein; mouse clone MAB377; Chemicon, Temecula, CA, USA; 1:2000). Signal was detected using the chromogen 3-amino-9-ethylcarbazole (Sigma-Aldrich, St. Louis, MO, USA).
RNA isolation
For RNA isolation, cells or frozen tissue were homogenized in Qiazol Lysis Reagent (Qiagen Benelux, Venlo, The Netherlands). Total RNA, including the miRNA fraction, was isolated using the miRNeasy Mini kit (Qiagen Benelux, Venlo, The Netherlands) according to manufacturer’s instructions. The concentration and purity of RNA were determined using a Nanodrop spectrophotometer (ThermoFisher Scientific, Waltham, Massachusetts, USA). FFPE material was processed for RNA isolation using QuickExtractTM FFPE RNA Extraction Kit (Epicentre, Madison, WI, USA) according to manufacturer’s instructions. The concentration of RNA was determined using a Qubit® 2.0 Fluorometer (Life Technologies, Carlsbad, CA, USA).
MicroRNA microarrays
A screening for miRNAs was performed using the miRCURY LNATM microRNA array (7th gen, Cat # 208500, Exiqon, Vedbaek, Denmark) by the Exiqon miRNA array service. Briefly, 5 μg of total RNA from 6 GG and 5 control cortex samples were labelled using the miRCURY LNATM microRNA Hi-Power Labeling Kit, Hy3TM/Hy5TM and hybridized on the miRCURY LNATM
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