For differentiation of LUHMES cells towards dopaminergic neurons, cells were cultured in advanced DMEM/F12 (Life Technologies GmbH, Darmstadt, Germany) supplemented with 1% N2-Supplement, 2 mM L‑glutamine, 1 mM dibutyryl cAMP, 2 ng/mL GDNF and 1 μg/mL tetracycline. After 48 hours, cells were trypsinized and seeded with 7.5 x 104 cell/cm² in pre-coated flasks. Every 48 hours after re-seeding, fresh differentiation medium was added to the cells for six days. To induce a PD-like phenotype, LUHMES cells were treated with 10 µM 1-methyl-4-phenylpyridinium (MPP+; Sigma Aldrich, Munich, Germany) 6 days after initiation of differentiation for 48 hours. Control cells were supplemented with H2O.
Growth protocol
LUHMES cells were cultured in flasks pre-coated with 50 µg/mL Poly-L-ornithin and 1 µg/mL Fibronectin. Cells were cultured in advanced DMEM/F12 (Life Technologies GmbH, Darmstadt, Germany) supplemented with 1% N2-Supplement, 2 mM L-glutamine and 40 ng/mL basic fibroblast growth factor.
Extracted molecule
total RNA
Extraction protocol
Total RNA including miRNAs was isolated using the Qiagen miRNeasy Kit according to manufacturers recommendations.
Label
Cyanine 3
Label protocol
The expression profile of 2549 human miRNAs was determined using Agilent miRNA Complete Labeling and Hyb Kit (Agilent, Cat. No 5190-0456) according to the manufacturers recommendations
Hybridization protocol
The expression profile of 2549 human miRNAs was determined using SurePrint G3 8x60k miRNA microarrays (Agilent) according to the manufacturers recommendations
Scan protocol
Arrays were scanned with the Agilent Scanner with default settings.
Description
miRNA expression after MPP+ treatment of LUHMES cells
Data processing
Analysis of the received fluorescence signals was done by using Agilent AGW Feature Extraction software (version 10.7.1.1, Agilent Technologies, Santa Clara, CA, USA). Normalization of background corrected values was performed using GeneSpring by biological significance analysis (version 14.9, Agilent Technologies, Santa Clara, CA, USA).
