We excluded Wnt-3α because it exhibited relatively stable expression irrespective of the incubation conditions, and another three genes (AP-1, GSK-3β, and CTNNB) were excluded because their expression levels were not significantly altered by inflammatory factors.
Some of the genes located within CNV regions were not included in the analysis because their expression levels were below the average of negative control genes included in the array (bacterial genes lysA, pheA, thrB and trpF) across the different experiments (6.139), indicating that the hybridization to the array probes is below the level of detection.
Expression of 15 of the remaining 17 candidates was not clearly detected in any brain region by in situ hybridization, possibly because their expression levels were below the detection threshold, whereas the expression of the remaining 2 candidates was detected in both the OLs and other brain regions in the honeybee brain, suggesting that these genes represented 'false positive clones'.
In addition, it is important to note that other genes were contained within the identified CNVs, but were not included in our analysis because their expression levels were not reported in the above-mentioned study or because the MLPA probes we used did not coincide exactly with the gene.
While native PKA-RI subunits could potentially act as endogenous high affinity soluble cAMP buffers, free regulatory subunits are rarely found in the living cell because their expression levels are tightly controlled in a 1∶1 ratio with those of PKA-C [27], [28].
miRNAs whose normalized expression of two libraries is smaller than one were removed, because their expression levels are too low.
We excluded from the analysis probes that were non informative either because their expression level was below detection or because their expression level did not change (>2 folds) along the experiments.
Two of the analyzed predicted miRNAs (miR901T and miR904T) did not show detectable hybridization signals, probably because their expression level was low (isolation frequency < 25).
Future studies should aim to go beyond analyzing Bcl-2 family protein levels because their expression level often poorly indicates protein activation, especially given that most BH3-only members undergo strong regulation by post-translational mechanisms.
However, for miR395 and miR399 low expression is not surprising because their expression level is generally low under normal growth conditions as they are induced by sulphur or phosphate deficiency respectively [ 21, 22].
The expression levels of all putative transcripts were calculated and 19,465 putative transcripts were discarded as potential pre-mRNAs because their expression level fell below 5% of the major isoform of the corresponding gene [ 34].
