Sample 29_neg
Homo sapiens
Protocols: Surgical sampling: 5ALA was administered orally to patients 2-4 hours prior to surgery at 20mg/kg dose. All patients underwent craniotomy with intra-operative image guidance and visualization of 5ALA induced fluorescence using an appropriately equipped operative microscope (Leica OH-X). Multi-region sampling was conducted, and sample location determined using image guidance. Samples were taken from non-fluorescent or minimally fluorescent tumor core (corresponding to T1 with gadolinium non-enhancing or heterogeneously enhancing central tumor), viable fluorescent tumor rim (corresponding to peripheral strongly gadolinium enhanced areas on T1 MRI) and from the areas of 5ALA induced fluorescence furthest into the area of MRI T2 high signal beyond the bulk tumor, where tumor blended into brain in an invasive fashion (non-enhancing on T1 with gadolinium). Samples from each region were subdivided into specimens for snap freezing in liquid nitrogen, formalin fixation and paraffin embedding for histology and for enzymatic dissociation for FACS analysis. Histological diagnosis of glioblastoma was confirmed by intra-operative smear and formal post-operative diagnosis (including IDH mutations, ATRX mutation and MGMT methylation status) by the regional brain tumor neuropathology service. Fluorescence Activated Cell Sorting (FACS): Cells were sorted using the MoFlo XPD sorter (Beckman Coulter) with excitation at 405 nm and emission detection at 605 - 625 nm. For increased purity, cells were sorted twice consecutively. U251 GBM cells incubated for 2 hours with and without 5-ALA were used as controls to set gates for sorting. The positive and negative sorted cells were centrifuged at 800 rpm (180×g) for 5 minutes at room temperature and cell pellets snap frozen. mirVANA RNA isolation kit NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB: E7490), the NEBNext Ultra Directional Library Kit for Illumina (NEB: E7420) and the NEBNext Multiplex Oligos for Illumina (Index Primers Set 1) (NEB: E7335L).