BMC Genomics 2006, 7:252 http://www.biomedcentral.com/1471-2164/7/252 FAi,geuxrpeer1imental design A, experimental design. B&C&D, correlation of log2 intensities within and between protocol groups. B&C, scat- ter plots of log2 signal intensities from a pair of replicates treated with the OneRA and the TwoRA protocol respectively. D, scatter plot of log2 signal intensities averaged across the OneRA replicates versus that from the TwoRA replicates. The coeffi- cient of correlation (r) value is given for each pair. Similar results were obtained with the SN and SA sample groups. To test whether protocol variability is significantly greater than the residual variability, we derived p-values from the F-values (MSA/MSE) for each gene (using the upper tail of an F-distribution with 1 and 3 + 4 – 2 degrees of freedom). In fact, the p-values were far from uniformly distributed. Storey suggests the following estimate of the proportion of hypotheses from the using p-values: the fraction of p-values above the median p-value m, divided by (1-m) [38]. This results in the following estimate of the propor- tion of genes with significantly higher amplification vari- ability: DRG 47%, SN 50%, SA 41%. That is, in all cases at least 40% of genes show differences between protocols, which are not explained by variability within replicates. Signal distortion following TwoRA To investigate signal distortion by the TwoRA protocol, we looked at the well-documented 3' bias feature of the pro- tocol in the DRG dataset (similar results were obtained with the SA and SN). Bias arises from greater 3' represen- tation of targets during TwoRA while 5' regions are often lost. This causes amplified RNA to be shorter than normal. Previously published work explored the increase in the 3'/ 5' signal ratios only from control genes (GAPDH, Actin) to prove that such effect occurred)[2,4,7]. In this study, we undertook a more comprehensive analysis by using infor- mation from all probesets on the array. More specifically, we correlated the deviation in log2 intensity following TwoRA (Δlog2IN) – obtained by subtracting the log2 intensity in the OneRA from that of the TwoRA – with the probesets 3' locations on corresponding targets (see meth- ods for a description of how these locations were obtained) (Figure 2). The trend suggests that probesets distal from the 3' end are more likely to endure an atten- uation of signal intensity following TwoRA whilst those close to the 3' end are likely to show intensification of sig- nal. Page 3 of 14 (page number not for citation purposes)