Combining probe sets that were either significantly up-regulated (0.999) or significantly down-regulated (0.001) in at least one of the comparisons (EvN, SvN and SvE) generated a list of 409 probe sets.
These microarrays were the Qiagen-NRSP8 commercial array [ 17] and a microarray we constructed, referred to as SLA/PrV, which combines probe sets specific to genes localized in the SLA complex, genes encoding other important immunological molecules [ 18] and all the PrV genes.
The resulting data sets comprised 5 columns: combined probe IDs, gene name, gene symbol, d score, and fold change.
When the 2 fold change and 1-way ANOVA filters were combined, 409 probe sets were selected: 366 (89%) of them up-regulated and 43 (11%) probe sets down-regulated.
Those clusters, which showed their highest expression either at Inv1, Inv2, Inv3 or Inv4 and showed no upregulation at earlier time points, were combined (379 probe sets).
After combining synonymous probe sets and removal of probes that did not correspond to a mapped gene, 24,754 genes were selected for further analyses.
The probes included for subsequent evaluation were combined into two probe sets.
Multiplex ligation-dependent probe amplification (MLPA) (Schouten et al., 2002) probes were designed according to the recommendations by Stern et al. (2004) and combined in different probe sets (Supplementary data, Table SII).
By combining the four probe sets (labeled with different fluorophores), this approach effectively quadrupled the number of probes that can be used in an available size range.
Quantitative real-time PCR was carried out using the predesigned FAM MGB combined primer and the probe sets termed assays on demand for NGF, BDNF and substance P from Applied Biosystems (Warrington, UK): NGF, Hs00171458_m1; BDNF, Hs00542425_s1; substance P, Hs00243225_m1.
