8 protocols
The data were normalised using q-quantile normalisation with BASE (BioArray Software Environment) application. BASE is described in Vallon-Christersson J, Nordborg N, Svensson M, Häkkinen J. BMC Bioinformatics 2009, 10:330. doi:10.1186/1471-2105-10-330.
Standard Illumina scanning protocol
ID_REF = VALUE = q-quantile normalized
Standard Illumina hybridization protocol
Biotinylated cRNA were prepared with the Illumina® TotalPrep™ RNA Amplification Kit.
U87 MG glioblastoma cells were grown at normoxic (21% oxygen) or hypoxic (1% oxygen) conditions for 48 hours. Conditioned media from normoxic and hypoxic cells were then used to isolate exosomes by differential centrifugation. Both cells and exosomes were lysed in Trizol reagent and RNA was isolated.
RNA was extracted with Trizol reagent, followed by DNase I treatment with DNA-free™ Kit (Ambion) in accordance with the protocol provided with the kit. Quality control was performed with Agilent Bioanalyser.
Routine culture medium: High Glucose DMEM supplemented with 10% FBS, 2 mM L-glutamine and 100 U/ml penicillin and 100 µg/ml streptomycin. Experiment medium: High glucose DMEM supplemented with 1% BSA, 2 mM L-glutamine and 100 U/ml penicillin and 100 µg/ml streptomycin. Routine culture was done in a humidified incubator maintained at 37 °C with 5% CO2 and 95% air. For hypoxia experiments, cells were incubated in a humidified 5% CO2 InVivo2 Hypoxia Work station 400 (Ruskinn Technology Ltd) set at 37 ºC and 1% O2.