Although many studies have used microarray technology for expression profiling, the criteria for determining putatively regulated genes from such analyses remain poorly defined, the current standard being a >2-fold difference in replicate arrays. Using Affymetrix high-density rat oligonucleotide-array data from control (naïve, non-injured) and experimental (axotomy, 3 day post peripheral nerve injury) DRGs, and comparing the differences detected in control vs. control and control vs. experimental arrays, we have explored the optimal criteria for minimizing the false positive error rate. A >2-fold criterion alone was found to generate a substantial proportion of potential false positives, while also excluding many putatively regulated genes. Based on significant difference for each probe set (P < 0.05), 109 genes differed in the control vs. control group, and 556 in the control vs. experimental group, again a prohibitively high false positive rate (20%). However, a combination of fold and significant difference was found to maximize the difference between control vs. control and control vs. experimental array comparisons. Using >1.5-fold and P < 0.05, 240 putatively regulated genes were detected in the experimental group compared with only 12 in the control group, an estimated error rate of 5%. If fold differences of 1.25 to 1.5 are included, many more significantly different genes are recruited (219 in the experimental vs. 41 in the control comparison) reducing the potential false negative rate. At the same time, the estimated false positive error rate rises to 11.5 %, still lower than the 2-fold alone error. Producing a reduction in false positive without also producing false negative differences requires the use both of fold and significant difference. The established 2-fold criterion is much too conservative; when using replicate arrays with pooled samples from multiple animals to reduce biological variation, it loses over 50% of putatively regulated genes and fails to eliminate false positives. The very high concordance rate found between genes predicted from the array analysis to be regulated and measurements of mRNA for 24 genes by quantitative Northern slot blots and in situ hybridization, validates utilization of a two dimensional matrix of fold and significant difference.
One problem with oligonucleotide arrays is sensitivity. Some groups of transcripts, particularly GPCRs, including the opiate receptors, which are expressed in the DRG,  fall below the microarray detection threshold. Lack of sensitivity may result from technical issues such as poor probe performance  or low copy number transcripts. Tissue heterogeneity and neuronal subpopulation-restricted gene expression may lower the concentration of a transcript in the total RNA sample to below detection threshold .
Recently Xiao et al  published a study using 7.5 K cDNA arrays to analyze genes regulated in the DRG 2, 7, 14 and 28 days following sciatic nerve axotomy. Of the genes listed in Table 4, seven were found in both studies (Peripheral Benzodiazepine receptor, 5HT3, Gadd45, CLP36, Lysozyme, SNAP25 and VGF). Of those genes whose expression was analyzed across time in this study (Figure 5) two, Lysozyme and SNAP25, were also detected by Xiao et al . Both genes show very similar expression patterns over time in the two studies.
In a different cDNA array study, also recently published, 16 genes were identified to be regulated by at least 2-fold one week after sciatic axotomy . Of these, seven were also identified by our study. Five of the non-concordant genes were not represented by known probe sets on the array used here. Others changed, but did not reach statistical significance, and likely represented differences in regulation levels relative to the time points used (1 week as opposed to 3 days). One gene verified in this study, the small proline-rich protein 1A (Table 2), was characterized in detail by Bonilla et al, who show that it colocalizes with filamentous actin in membrane ruffles and augments axonal outgrowth .
Comparison of the global lists of regulated genes between this study and other reports are complicated by the differing criteria used to identify regulated genes in the different studies. However, it is encouraging to note that a reasonable number of genes have been cross identified in three separate array studies looking at the effects of peripheral nerve injury on dorsal root ganglion gene expression, pointing to the reliability of array technologies across differing platforms.
Why is the number of genes regulated in sensory neurons after peripheral nerve injury so large? The injury constitutes an enormous stress to the cell and also deprives it from contact with its normal target-derived neurotrophic support. Both of these events represent perturbations from normal cellular functioning that require a variety of compensatory responses. A major issue is cell survival. Injured adult DRG neurons do not die after axonal injury  as a result of the upregulation of cell survival factors such as the small heat shock protein HSP27 . We now find several genes that could have a survival-promoting role, including the peripheral benzodiazepine receptor, whose agonists are potent antiapoptotic compounds , GADD45A, which maintains genomic stability and appears to be anti-apoptotic in neurons after ischemic damage , metallothionein 1L, which is a free radical scavenger , GTP cyclohydrolase 1, which is obligatory for the actions of NGF on PC12 cells , and IES-JE (MCP-1) which attracts macrophages, which in turn have been reported to have a role in neuronal survival .
Neurons with axons in the peripheral nervous system can successfully regenerate when injured . This is in part due to an environment permissive for axonal growth but also due to the upregulation of regeneration-associated genes (RAGs) which increase intrinsic growth capacity . Three examples of RAGs are GAP-43 and CAP-23  and sprp1A . We now find several other potential candidates including; α2 macroglobulin, CLP36, and VGF all of which have been described to promote neurite outgrowth or interact with the cytoskeleton in a way that may promote regeneration (see Table 4 [additional file 3] for references). MMP3 also known as 53 kD polypeptide, transin or stromelysin-1, a secreted protease, may facilitate neurite growth by dissolving the extracellular matrix of the basal lamina at the growing tip of the axon (see Table 4 for references).
Sensory neurons react to peripheral nerve injury by increasing their excitability, changing their constitutive synaptic transmitter profile and even their synaptic contacts in the spinal cord. Interestingly, an analysis of the functional classes of the genes differentially expressed (Figure 6) reveals the contribution that alterations in transcription make to these processes. Several of these changes are maladaptive in the sense that they contribute to the generation of the abnormal sensations that constitute neuropathic pain, by producing ectopic spontaneous activity into the CNS, altering synaptic drive, increasing excitability and diminishing inhibitory action in the dorsal horn of the spinal cord, and by making novel synaptic contacts with inappropriate neurons [1, 4]. A number of the injury-regulated genes we describe here may contribute directly to this altered sensory processing; CB1 receptor, VGF, the phospholemman ion channel, SNAP25 A, endothelin-1 and the ligand-gated 5-HT3 receptor (Table 4, see additional file 3). We have only validated a small fraction of the putatively regulated genes detected by the arrays. Our analysis indicates that at a conservative estimate over two hundred genes (240 genes, >1.5-fold, P < 0.05) are regulated by the injury. These include genes of a number of distinct functional classes, some of which appear to change in a uniform way. Ion channels and neurotransmitter related genes tend to decrease their expression while those associated with the cytoskeleton increase. This raises the possibility that numbers of different genes may be regulated together by common changes in signal transduction and transcription induced by the injury, and contribute in this way to the coordinated changes that constitute the axotomy response.
Discovering and validating regulated genes by array analysis is clearly only the first step in elucidating their functional role. An evaluation of the cellular localization and distribution of the gene product as well as loss and gain of function manipulations in vitro and in vivo are required. Functional studies cannot yet be performed at a high throughput level and this represents one of the major limiting factors in analyzing the large numbers of candidate genes identified by microarray analysis. Elucidating the specific role of genes regulated in sensory neurons after peripheral nerve injury will provide insight though into many major biological issues including cell survival, growth, intercellular communication and the factors that contribute to sensory abnormalities. Microarray technology provides a powerful tool for beginning this analysis in a high throughput mode by revealing the extent of change in neuronal gene expression. Such analysis requires replicate measures to minimize variability, calculation of fold and significant differences to detect regulated genes with minimal false positive and negative confounders and validation of individual genes.