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Genes regulated by FGF2 in mouse cortical neuron culture

ABSTRACT: We have performed the hybridisation in triplicate using total RNA samples pooled from three independent batches of primary cortical neuron culture derived from E14.5 CD1 mouse brains on one day in vitro (1DIV). We stimulated the cells with 50 ng/ml FGF2 in the presence of 10 μg/ml heparin for 4 hours, and the expression of genes were compared to those in the absence of FGF2. The data were obtained from the three experiments with the best possible conditions and strict measures were applied for the purpose of quality control. Seventeen features representing housekeeping genes in the array (GAPDH, HPRT and S16, S9, and S8 ribosomal proteins) gave average value of Ratio of Medians (ROM), 1.20 ± 0.28, which was confirmed to be acceptable. Total RNA was extracted from cells in the growing phase by using RNA Clean (Hybaid) according to the manufacturer’s instruction and further purified with RNeasy mini kit (Qiagen). Direct labelling of 20 µg of total RNA primed with Oligo(dT) was performed in the presence of Cy-5 and Cy-3-dUTP (Amersham Pharmacia Biotech) using SuperScript II (Invitrogen). The Cy5- and Cy3-labelled cDNA samples were then mixed and purified with GFX DNA purification kit (Amersham Pharmacia Biotech). The samples were added with 1.2 µg of salmon sperm DNA and 5 µg of Poly(dA) and dried in SpeedVac at 60°C. The high-density glass microarray chips were prepared at the EMBL core facility by spotting the PCR products prepared from the NIA 15k cDNA mouse clone set. Array slides were pre-hybridized in 6x SSC, 0.5% SDS, 1% BSA at 42°C for 1 hour and incubated in a boiled water for 2 min shortly prior to hybridisation. The cDNA samples were resuspended in 15 µl of hybridisation buffer (50% formamide, 6x SSC, 0.5% SDS and 5% Denhardt’s) and denatured by incubating at 95°C for 2 min. Hybridisation was at 42oC for 16 hours. The slides were washed for 10 min each with 2 x SSC, then with 0.5 x SSC, 0.1%SDS, followed by 0.1 x SSC, 0.1%SDS, at room temperature. Scanning was performed with GenePix 4000B (Axon). Quality control was performed, firstly by eye, to confirm scanner alignment and absence of significant bubbles and scratches. Scatter plots were further used to eliminate the unacceptable hybridisation data. The multiple spike-in controls RNA template were added to the sample upon direct labelling, and the successful labelling and hybridisation was confirmed in each hybridisations. GenePix Pro grogram calculates the Normalisation Factor of each hybridisation, based on the premise that the arithmetic mean of the ratios from every feature on the given array should be equal to 1. Normalisation was therefore performed by multiplying the Factor to Ratio of Medians (ROM) in each gene. The program also identifies features that did not give good alignment to the expected spotted area as “flagged” spots, indicative of the impaired-quality hybridisation of the specific genes. Keywords = NIA mouse 15k Keywords = cortical neuron Keywords = FGF Keywords: other

ORGANISM(S): Mus musculus

PROVIDER: GSE2066 | GEO | 2004/12/15

SECONDARY ACCESSION(S): PRJNA91317

REPOSITORIES: GEO

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Genes regulated by FGF2 in mouse cortical neuron culture

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