Driving neuronal differentiation through reversal of an ERK1/2-miR-124-SOX9 axis abrogates glioblastoma aggression, Sabelstrom et al
Description
Table S6. Heatmap showing semi-quantitative analysis of miRNAs with/without PD0325901 (1 uM) in patient-derived proneural GBM5 or classical GBM6 tumorsphere cultures after five days.
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Human GBM5 and GBM6 cells were plated at 1 x 104 cells/cm2 on polyornithine/laminin-coated plates in the presence of 1 uM PD0325901 or DMSO (control) for five days. Total RNA (5 ug) was isolated using RNeasy kit (Qiagen). Membrane-based miRNA array analysis was performed using Human miRNA Array III according to the manufacturer’s instructions (Signosis). In brief, miRNA were hybridized to two oligonucleotide primers to form a miRNA/oligonucleotide duplex and purified using magnetic beads. The purified RNA/DNA duplexes were then incubated with DNA ligase at 37°C for 90 min. After a brief extension of miRNA sequences using a PCR machine (Mastercycler, Eppendorf), T7 RNA polymerase was added to amplify the sequences. Membranes were pre-hybridized in 50 ml tubes and incubated with transcribed RNA at 42°C overnight in a hybridization oven. After washes in hybridization wash buffer, the membranes were rinsed in detection wash buffer, incubated with streptavidin-HRP conjugate for 45 min at room temperature, mixed with equal amounts substrate A and B, followed by ECL (Amersham).