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Transcriptional changes induced by rAAV-mediated Npas4 overexpression in hippocampal primary cell culture compared to GFP-control

ABSTRACT: Hippocampal neuron cultures were prepared from neonatal C57BL/6N mice as previously described (Bading and Greenberg, Science, 1991). Cells were plated in dishes coated with Poly-D-Lysine (2mg/ml) and Laminin (10 µg/ml). Two days prior to harvesting, the medium was changed from NBA to transfection medium. Infection with rAAV-Npas4-flag of rAAV-GFP-myc was performed on day 5 in culture and cells were harvested on day 11. Total RNA was isolated using the RNeasy kit (Qiagen GmbH, Germany) according to the manufacturer’s instructions, including on-column DNase I digestion. Microarray analysis was performed with Illumina MouseWG-6 v2.0 Expression BeadChips®. The Genomics and Proteomics Core Facility at the DKFZ (Heidelberg, Germany) processed the RNA samples. RNA quality assessment was performed with the Nanodrop ND-1000 (Agilent 2100 Bioanalyzer). 4 μg of RNA was used for each microarray replicate. Raw microarray data was analyzed using Chipster (Kallio et al., 2011) by using R and Bioconductor packages. Statistical testing was performed using t-tests over all beads (e.g., one probeID over all beads of all samples of a group) in original scale. Benjamini-Hochberg correction was applied over all p-values of the differential expression analysis. We used a false discovery rate (FDR) of 5 % as a cut-off for determination of significance.

ORGANISM(S): Mus musculus

SUBMITTER: Thomas Lissek

PROVIDER: E-MTAB-10706 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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