Kristiansen et al. BMC Genomics 2011, 12:551 http://www.biomedcentral.com/1471-2164/12/551 penicillin-streptomycin as described previously (SCG medium; [61]). To limit the proliferation of non-neuro- nal cells, the antimitotic agents fluorodeoxyuridine and uridine (Sigma Aldrich) were added to the SCG medium at a final concentration of 20 μM. For some experi- ments, 2.5S NGF (Cedarlane, Canada) was also added to SCG medium at a final concentration of 50 ng/ml. Neu- rons were plated on 13 mm diameter glass coverslips coated with poly-L-lysine and laminin placed in 3.5 cm diameter dishes containing 2 ml of SCG medium and NGF for 5-7 days. In NGF withdrawal experiments, neu- rons were washed twice in SCG medium lacking NGF and then refed with SCG medium supplemented with a neutralising anti-NGF antibody at 100 ng/ml (Chemicon Europe Ltd, UK). The MLK inhibitor, CEP-11004 (Cephalon, Inc., USA) was dissolved in DMSO and used at a final concentration of 400 nM. RNA extraction Total RNA was isolated from sympathetic neurons cul- tured for 7 days using an RNeasy mini kit (Qiagen). An on-column DNase digestion was performed to eliminate genomic DNA contamination using DNase I according to the manufacturer’s instructions (Qiagen). RNA con- centrations were determined using a NanoDrop spectro- photometer (ND-1000). RNA was further analysed for integrity and quality on an Agilent Bioanalyser. Array hybridisation Up to 2 μg of total RNA was processed and labelled using the Affymetrix GeneChip Whole Transcript Sense Target Labelling Assay as outlined in the manufacturer’s instructions. Hybridisation to Affymetrix Rat Exon 1.0 ST arrays was performed for 16 hours at 45°C with con- stant rotation. Exon array data are available from the ArrayExpress database (http://www.ebi.ac.uk/arrayex- press) under accession number E-MTAB-696. Analysis of array data Signal estimates and normalisation for gene-level ana- lysis were generated using the Probe Logarithmic Intensity Error Estimation (PLIER) algorithm imple- mented in the Expression Console software (Affyme- trix). Only core, non cross-hybridising probe sets that map to well-annotated exons were included. To reduce noise, probe sets and transcript clusters which fell into the lowest quartile of the expression signal distribution across all samples were excluded from the dataset. Sig- nal values were analysed using Bioconductor (R). Gene expression values were compared between the three sample groups (+NGF, -NGF and -NGF+CEP-11004) using the moderated t-statistic of the Bioconductor package, Limma [62]. To correct for multiple testing at the gene level, the Benjamini-Hochberg (FDR) test was Page 14 of 17 applied to identify statistically significant differentially expressed genes (FDR adjusted p-values < 0.05). Lists of significantly up- and down-regulated genes obtained from statistical comparisons were subjected to func- tional enrichment analysis using DAVID annotation tools [63]. Real-time quantitative PCR Up to 1 μg of total RNA was reverse transcribed into cDNA using SuperScript II reverse transcriptase (Invi- trogen) and oligo(dT) as described previously [64]. Ten nanograms of cDNA template was used for real time quantitative PCR using the ABI-Prism 7900HT fast Sequence Detection System (Applied Biosystems, War- rington, UK). Txnip, ddit3, trib3, ndrg1 and mxi1 mRNA levels were normalised to the level of the control genes, gapdh or hprt1. Pre-optimised Taqman® gapdh (Rn99999916_s1), hprt1 (Mn00446968_m1), txnip (Rn01533885_g1), trib3 (Rn00595314_m1), ddit3 (Rn00492098_g1), ndrg1 (Rn01506130_m1), and mxi1 (Rn00565846_m1) primer/probe sets were supplied by Applied Biosystems. QPCR reactions were set up with 1 × primer/probe set and 1 × Taqman® PCR Master Mix (Applied Biosystems). PCR conditions were 95°C for 20 seconds, followed by 40 cycles of 95°C for 1 second and 60°C for 20 seconds. QPCR data was analysed using the 2-ΔΔCT relative quantitation method [65]. Immunoblotting Immunoblotting was performed as described previously [24]. Briefly, sympathetic neurons were harvested in 1 ml of ice-cold PBS, spun down and lysed in sample buf- fer (2% SDS, 2 mM b-mercaptoethanol, 60 mM Tris, pH 6.8, 0.01% bromophenol blue) for 10 minutes at 100°C. Proteins were separated on 12% SDS polyacryla- mide gels and transferred to Immobilon-P (Millipore). After blocking for 45 min with 5% non-fat milk in TBS supplemented with 0.5% Tween-20, the membrane was incubated with different primary antibodies overnight at 4°C. The following primary antibodies were used: rabbit polyclonal Trib3 antibody (M-165; Santa Cruz, USA), rabbit polyclonal Ndrg1 antibody (H-60; Santa Cruz), mouse monoclonal Txnip antibody (MBL International, Woburn, USA), mouse monoclonal CHOP10/Ddit3 anti- body (B3; Santa Cruz), rabbit polyclonal Mxi1 antibody (G16; Santa Cruz), mouse monoclonal c-Jun antibody (BD Transduction Laboratories). Equivalent protein loading was confirmed by using a rabbit polyclonal ERK 1/2 antibody (Cell Signalling Technology). Immunofluorescence Sympathetic neurons cultured on poly-L-lysine/laminin- coated glass coverslips were fixed using 4%