Figure 5

Validation of gene induction after NGF withdrawal by real time PCR. Rat sympathetic neurons were cultured in vitro for 6 days and then maintained for 16 hours in 1) medium containing NGF, or 2) medium without NGF supplemented with anti-NGF antibody or 3) medium without NGF containing anti-NGF antibody and the compound CEP-11004 at 400 nM. RNA was then isolated using an RNeasy kit. The experiment was performed in triplicate and the resulting RNA samples were then processed and an Affymetrix rat exon microarray analysis was carried out. A, The fold increase in RNA level after NGF withdrawal or after NGF withdrawal in the presence of CEP-11004 determined by exon array analysis (average of 3 independent experiments) is shown for 5 representative genes. For each gene, the level of expression in the presence of NGF was set as 1 and the expression levels for the other treatments were normalised relative to this. B-C, mRNA levels for the 5 genes were also measured by real time PCR using RNA prepared from three independent sets of neurons and normalised to the housekeeping genes gapdh (B) or hprt1 (C). The data shown represents the average of three independent experiments ± SEM. Statistical comparisons were made between +NGF and -NGF or -NGF and +CEP-11004 for each gene. *p < 0.01.