BMC Genomics 2006, 7:252 http://www.biomedcentral.com/1471-2164/7/252 protocol on ratios, a larger profile of differential expres- sion is needed. This was possible with the (DRG,SN) pair. Thus, we decided to base our assessment of the effect of the TwoRA on ratios on the (DRG,SN) dataset. other sample is minimally reduced (Figure 6a &6c). Less frequently, the intensity increases with amplification in one of the samples but not in the other sample (Figure 6b &6d). Encouragingly, log2 expression ratios from the (DRG,SN) treated with the OneRA and the TwoRA protocols are comparable; though they show more variability than their counterparts from the (SA,SN) pair (Figure 5c). Moreover, the regression line (shown in blue) appears to be shifted from the diagonal in a way that suggests that the expres- sion ratios are on average slightly lower in the TwoRA rel- ative to the OneRA. Variation in ratios From our previous analysis, we know that the TwoRA pro- tocol may shift the absolute intensity levels. However, this only affects expression ratios if the intensity is shifted une- qually in the two biological samples. That is, deviations in intensity (Δlog2IN) following TwoRA, that differ in the two samples, can result in variability in the expression ratios from the OneRA and the TwoRA groups. To get further insights into how unequal shifts in the intensity level following amplification of different biolog- ical samples affect the expression ratios, we ranked probesets by the absolute difference in their OneRA and TwoRA log2 expression ratios and selected the top 100 for further analysis (partly listed in Table 1). Specifically, we examined the intensities of these selected probesets across all four groups: the OneRA and the TwoRA DRG, SN. The resulting intensity profiles were classified into four catego- ries depending on the direction of change in intensity and the tissue where this change occurred (Figure 6). The most populated categories show a significant reduction in the intensity in one of the samples whilst the intensity in the ProbeSet ID (Gene Symbol) 1425983_x_at (Hipk2) 1422277_at (Glra1) 1422009_at (Atp1b2) 1450177_at (Ngfr) 1425337_at (Slc12a5) 1425963_at (Cabp7) 1439083_at (Ahi1) 1429566_a_at (Hipk2) 1456637_at (Lrrtm2) 1423669_at (Col1a1) Interestingly, with all four categories, expression ratios are reduced following TwoRA. Moreover, the majority of the selected probesets have varying intensity levels in the DRG versus SN, OneRA. Frequently these genes are absent in one sample but highly expressed in the other sample (shown as coloured lines in figure 6), which may explain the deviation in expression ratios following TwoRA. If one takes the example of HipK2, the log2 intensity in the SN was reduced from 8.20 in the OneRA to 0.73 in the TwoRA. However, HipK2 is absent in the DRG (the OneRA log2 intensity is 0.87), thus an equivalent reduc- tion in the intensity level in this sample is not possible (floor effect). As such the log2 expression ratio is shifted from -7.33 in the OneRA to 0.15 in the TwoRA. Alterna- tively, in other cases, if amplification increases the inten- sity in one sample, an equal shift in the other sample would not be possible if the intensity was close to satura- tion (ceiling effect). Thus, distortions in the expression ratios may occur when a shift in intensity (Δlog2IN) in one sample cannot be mirrored in the other sample because it would cause the intensity to fall outside the dynamic range of the scanner. This relates to the previous analysis presented in Figure 4. Certainly, this phenomenon does not apply to genes with similar intensity levels in the two samples since a shift in intensity in one sample is equally possible in the other sample. To assess the extent to which this phenomenon explains the deviation in expression ratios observed across the 1R 0.87 2.35 2.98 8.98 2.34 1.94 2.14 1.62 8.57 11.06 DRG 2R 0.89 2.40 2.56 2.63 2.33 1.98 2.17 1.55 3.35 5.86 Log 2 intensity SN 1R 8.20 9.65 12.73 2.25 9.89 7.88 7.77 11.01 3.92 2.76 2R 0.73 2.46 5.50 2.33 3.50 2.07 2.05 5.60 4.00 2.81 1R 7.79 9.87 12.57 2.23 9.63 7.80 7.96 10.78 4.59 2.74 SA 2R 0.70 2.46 4.49 2.27 3.54 2.03 2.54 5.71 4.07 2.74 Table 1: Summary of the top 10 discrepant probesets whose (DRG,SN) expression ratios from the TwoRA deviate most from the OneRA. Listed are gene annotations (probeset ID and gene symbol) and log2 intensities from all biological versus protocol groups. Page 8 of 14 (page number not for citation purposes)