BMC Genomics 2006, 7:252 http://www.biomedcentral.com/1471-2164/7/252 implies that among these deviant probesets, there is a population that is at the same distance from the 3' end as the general population; yet, their intensities are shifted in the TwoRA. This suggests that other factors, apart from 3'bias, are likely to be involved in distorting the signal. One possible factor is the abundance of the transcript. Thus, we correlated the deviation in log2 intensity follow- ing amplification (Δlog2IN) with the log2 intensity for the DRG, OneRA (Figure 4). The plot indicates some bias in the extreme Δlog2IN values along the intensity scale. This is due to the fact that the intensity range is limited; in other words, intensity cannot be higher than the satura- tion level (ceiling effect) or below background noise (floor effect). Fidelity in maintaining expression ratios The ultimate aim of microarrays is the identification of differential expression. Thus, a good amplification proto- col should faithfully maintain expression ratios. To verify this, we cross-compared expression ratios from biologi- cally distinct tissue samples treated with the OneRA and the TwoRA protocols. First, we considered the (SA,SN) pair. Expression ratios on log2 scale from the OneRA samples were correlated with their equivalents from the TwoRA (Figure 5a). The signif- icant changes in expression (labelled in figure 5a) seem to be consistent in the TwoRA and the OneRA groups. These are well documented in the literature (activating transcrip- tion factor 3 [19] and small proline-rich repeat protein 1A [20]). However, there are relatively few differences in gene expression between these two biological samples, proba- bly due to the fact that the tissue from the injured animals included areas of the spinal cord not affected by the axot- omy, which could have caused a dilution of effect in the relevant areas. To reliably evaluate the effect of the TwoRA DFiegvuiarteio4n in log2 intensity following TwoRA (Δlog2IN) versus the OneRA log2 intensity Deviation in log2 intensity following TwoRA (Δlog2IN) versus the OneRA log2 intensity. The dashed lines in red show roughly the limits of the intensity range, thus the upper line represents saturation while the lower line represents back- ground noise. The analysis was done on the DRG data; similar results were obtained with the SN and SA samples. Page 6 of 14 (page number not for citation purposes)