BMC Genomics 2006, 7:252 http://www.biomedcentral.com/1471-2164/7/252 Since the selected probesets suffer from a floor and ceiling effect, we shall refer to them as FCE probesets for the rest of the article. Clearly, the FCE probesets are the same probesets that show the most deviant expression ratios following TwoRA (coloured in red, Figure 5c) and corre- spondingly the most pronounced variation in shifts in intensity (Δlog2IN) in the DRG and SN samples (in red, Figure 5d). In fact, the correlation in the (DRG,SN) expression ratios across protocols (r) = 0.89 is improved to 0.93 when the FCE probesets are excluded. Interest- ingly, we found that the FCE probesets show consistent Δlog2IN following TwoRA in the SA and SN groups (in red, Figure 5b). This is because their intensities are similar in the (SA,SN) but different in the (DRG,SN) datasets. Similarity in inferring significance in ratios The primary aim of a microarray experiment is to detect significant changes in gene expression. Our results suggest that ratios are most commonly reduced among the genes with large expression ratios in the OneRA, thus likely to be differentially expressed. Indeed, we found good evidence from the literature to suggest that 9 genes of those with the 10 most discrepant ratios following TwoRA are indeed dif- ferentially expressed between the SN and DRG (Table 2). Despite reductions in the ratios, genes can remain signifi- cant following TwoRA if their ratios are still large relative to the average population in the TwoRA. Moreover, among the population of genes with high expression ratios in the OneRA (Figure 5c), many do maintain their ratios in the TwoRA, most likely due to a faithful TwoRA of the transcripts in the two biological samples. Table 2: Evidence of differential expression for the genes with the 10 most discrepant (DRG,SN) tissue ratios from the OneRA and the TwoRA. ProbeSet ID Gene Symbol) Log2 expression ratios Evidence for differential expression OneRA TwoRA 1425983_x_at (Hipk2) -7.33 0.15 1429566_a_at (Hipk2) 1422277_at (Glra1) 1422009_at (Atp1b2) 1450177_at (Ngfr) 1425337_at (Slc12a5) 1425963_at (Cabp7) 1439083_at (Ahi1) 1456637_at (Lrrtm2) 1423669_at (Col1a1) -9.40 -7.29 -9.75 6.73 -7.54 -5.95 -5.63 4.65 8.30 -4.06 -0.06 2.94 0.30 1.16 -0.09 0.12 -0.65 3.05 Hipk2 encodes Homeodomain-interacting protein kinase 2. On the chip, there are two interrogation sites and in both the results from the OneRA indicates that the gene is more strongly expressed in the spinal cord. In the embryo, Hipk2 is expressed in DRG and spinal cord [24] but it is strongly downregulated prenatally in sensory neurons [25]. Judging from LacZ expression in transgenic mice, Hipk2 is highly expressed in the spinal cord, especially in the motor neurons [26]. Glra1 encodes the alpha1 subunit of the glycine receptor. Although it is found in some neurons of the DRG, it is very strongly expressed in the spinal cord [27]. ATP1b2 encodes the beta2 subunit of the Na+/K+ transporting ATPase, which is mainly located in glial cells [28]. It is not found in the DRG using immunohistochemistry [29]. Ngfr encodes the low affinity neurotrophin receptor p75. In the adult it is expressed in many neurons of the DRG [30], but largely absent in the spinal cord [31]. A second probeset (ID: 1421241_at) interrogating the same gene reported similar results i.e. the log2 expression ratio in the TwoRA is reduced (3.62, 0.22 OneRA, TwoRA respectively). Slc12a5 is the Solute carrier family 12 member 5, also known as the Neuronal K-Cl cotransporter 2 (KCC2). It is strongly expressed in neurons of the adult CNS including the spinal cord but it is absent in DRG neurons [32]. Cabp7 encodes the calcium binding protein 7, which is strongly expressed in spinal cord [33]. Ahi1, Abelson helper integration site 1, encodes a cytoplasmic adaptor protein [34]. It is primarily expressed in the CNS including the spinal cord [35]. Mutation of this gene causes Joubert syndrome, a congenital malformation of the cerebellum and brainstem with axonal decussating abnormalities of the corticospinal tract [35]. Lrrtm2 is leucine-reach repeat protein 2, also known as neuronal leucine-rich repeat protein 2. It is found in DRG and spinal cord [36] and on the basis of available in situ hybridisation data appears to have stronger expression in the DRG than in spinal cord [36]. Col1a1, is collagen type 1. It is ubiquitously expressed, but prominent in bone and connective tissue. It is possible that the increase in the DRG sample is due to a contamination during the dissection of the DRG (which were harvested after cutting the vertebral column). The genes have large expression ratios in the OneRA that are reduced in the TwoRA. The log2 expression ratios are calculated as log2 intensity from the DRG group – log2 intensity from the SN group. Thus, if the ratio is positive, the gene is more expressed in the DRG whilst if negative, the gene is more expressed in the SN. Page 10 of 14 (page number not for citation purposes)