To restrict the proliferation of non neuro nal cells, the antimitotic agents fluorodeoxyuridine and uridine were added on the SCG medium at a ultimate concentration of 20 uM. For some experi ments, two. 5S NGF was also extra to SCG medium at a last concentration of 50 ng ml. Neu rons have been plated on 13 mm diameter glass coverslips coated with poly L lysine and laminin placed in three. 5 cm diameter dishes containing two ml of SCG medium and NGF for 5 seven days. In NGF withdrawal experiments, neu rons had been washed twice in SCG medium lacking NGF then refed with SCG medium supplemented by using a neutralising anti NGF antibody at a hundred ng ml. The MLK inhibitor, CEP 11004 was dissolved in DMSO and made use of at a ultimate concentration of 400 nM. RNA extraction Total RNA was isolated from sympathetic neurons cul tured for 7 days working with an RNeasy mini kit.
An on column DNase digestion was carried out to reduce genomic DNA contamination using DNase I according towards the suppliers guidelines. RNA con centrations had been established using a NanoDrop spectro photometer. RNA was additional analysed for integrity and quality on PD0325901 ic50 an Agilent Bioanalyser. Array hybridisation As much as two ug of complete RNA was processed and labelled employing the Affymetrix GeneChip Total Transcript Sense Target Labelling Assay as outlined inside the companies directions. Hybridisation to Affymetrix Rat Exon one. 0 ST arrays was performed for 16 hrs at 45 C with con stant rotation. Exon array information can be found in the ArrayExpress database underneath accession quantity E MTAB 696. Analysis of array data Signal estimates and normalisation for gene degree ana lysis had been created working with the Probe Logarithmic Intensity Error Estimation algorithm imple mented while in the Expression Console software package. Only core, non cross hybridising probe sets that map to very well annotated exons had been integrated.
To reduce noise, probe sets and transcript clusters which fell in to the lowest quartile of your expression signal distribution across all samples were excluded in the dataset. Sig nal values have been analysed utilizing Bioconductor. Gene expression values have been compared amongst the 3 sample groups employing the moderated t statistic on the Bioconductor package deal, A966492 Limma. To accurate for many testing at the gene degree, the Benjamini Hochberg test was applied to identify statistically considerable differentially expressed genes. Lists of drastically up and down regulated genes obtained from statistical comparisons have been subjected to func tional enrichment analysis using DAVID annotation resources. Authentic time quantitative PCR Up to 1 ug of total RNA was reverse transcribed into cDNA applying SuperScript II reverse transcriptase and oligo as described previously.