age: Gestation day 15 strain: Swiss Albino cell type: Murine primary cortical neurons age of culture: Day 6
Treatment protocol
No treatment
Growth protocol
Primary cultures of cortical neurons were established from E15 Swiss albino mouse brains. The cortices were dissected from E15 mouse embryos and washed with Hanks’ balanced salt solution (HBSS, 14025-092, Gibco, Invitrogen, USA). The cortical slices was dissociated with 0.05% (w/v) trypsin in HBSS without Ca2+/Mg2+ (14175-095, Gibco, Invitrogen, USA) for 30 min at 37 °C and neutralized with 1mg/ml trypsin inhibitor (T6522, Sigma, USA). Single cells were obtained by gentle trituration in Neurobasal medium (21103-049, Gibco, Invitrogen, USA) supplemented with B27 (17504-044, Invitrogen, USA), L-glutamine and Penicillin-streptomycin (Gibco, Invitrogen, USA). The cells were counted by trypan blue exclusion and seeded on to poly-d-lysine coated 24 well plates at a density of 120, 000 cells/cm2. Cultures were maintained at 37 °C with 5% CO2 in a tissue culture incubator. Cells were harvested on Days 2, 4, 6, 8.
Extracted molecule
total RNA
Extraction protocol
Total RNA (+ microRNA) was extracted from cells by a single-step method using Trizol (Invitrogen, Life Technologies, USA) according to the manufacturers’ protocol. The concentration and integrity of the RNA were determined using Nanodrop ND-2000c spectrophotometry (Nanodrop Tech, Rockland, Del) and denaturing polyacrylamide gel electrophoresis, respectively.
Label
Hy3
Label protocol
LNA-modified oligonucleotide (Exiqon, Denmark) probes for mouse miRNAs annotated in miRBase version 16.0 were used in the microarray. Total RNA (1 µg) was 3′-end –labeled with Hy3 dye using the miRCURY LNA™ Power Labeling Kit (Exiqon, Denmark).
Hybridization protocol
Hybridization on miRCURY LNA™ Arrays, using MAUI® hybridization system.
Scan protocol
Hybridization images were scanned and digitized using InnoScan 700 microarray scanner (Innopsys, France).
Data processing
miRNA microarray data were analyzed by subtracting the background signal (B532nm) from the test signal (F532nm).
