BMC Genomics 2006, 7:252 http://www.biomedcentral.com/1471-2164/7/252 We applied two different statistical tests (the Z-scores and limma) to identify transcripts differentially expressed in the (DRG,SN) tissue samples prepared with both proto- cols (One RA and TwoRA). With each statistical method, an FDR based multiple testing correction was used and genes were ranked by their FDR values in ascending order. To measure the extent of agreement between protocols over genes identified as significant, we defined the signifi- cance similarity score (or the SSS) as the proportion of genes common to subsets of highly ranked genes from the OneRA and the TwoRA (DRG,SN) comparisons. The results are summarised in Table 3. Higher SSS were obtained using limma compared to the Z-scores method. With limma, 87 out of the top 100 most significant genes were common to the OneRA and the TwoRA comparisons as oppose to 59 using Z-scores statistics. For a more global assessment of the effect of distortions in expression ratios on their statistical significance, we used a scatter plot of negated log p-values (nlPv) from the limma analysis of the OneRA and the TwoRA (DRG,SN) (Figure 7). The FCE probesets are highlighted in red on figure 7 and it can be seen that their nlPv are least corre- lated between the two protocols, due to distortions in the expression ratios (scatter on figure 5c). Amongst the FCE probesets, some still show reasonable nlPv following TwoRA (> 10). Inspection of these genes revealed that they have large expression ratios in the OneRA and mod- erate ratios in the TwoRA (the median log2 expression ratios was 5.09, 2.52 respectively). By contrast, those FCE probesets with low nlPv (< 10) in the TwoRA have had their log2 expression ratios reduced severely following TwoRA (median log2 ratio in the TwoRA = 0.43). Interestingly, the latter have, on average, moderate expres- sion ratios in the OneRA (median log2 ratio in the OneRA = 2.8). This is expected since with moderate expression ratios, any reduction would have a greater impact on their significance level. Indeed, looking at the whole popula- tion of probesets, out of those with a nlPv between 10 and 20 in the OneRA, only 69% have an nlPv above 10 in the TwoRA, compared to probesets with high nlPv (> 20) in the OneRA where 87% (in agreement with previous results, Table 3) of them have nlPv above 20 in the TwoRA. This suggests that the TwoRA protocol is more suitable with experiments where large differences in gene expression are occurring. Discussion Microarray technology is currently limited by the need for relatively large transcript quantities, which makes it inca- pable of handling small biological samples. The T7 in- vitro transcription has been widely explored to achieve a linear amplification of targets for microarrays. Much work has considered the effect of linear amplification on the ability to profile gene expression. Although, the reproduc- ibility of such techniques and their fidelity in maintaining absolute levels of expression have been extensively ana- lysed, much less is known about their ability to accurately reproduce differential expression in distinct biological samples. This study gives further insights on the impact of linear amplification using the Affymetrix small sample protocol on expression ratios and differential expression. Our analysis confirms the high reproducibility of the small sample TwoRA protocol and the occasional failure in its fidelity to maintain the original levels of gene expres- sion. In this study, robust analyses were used to confirm the 3' bias role in signal distortion. Instead of limiting our analysis to control genes, as previously undertaken by other groups [2,4,6,7], evidence was obtained by exami- nation of intensity data from all probesets on the array. In addition to 3'bias, this work has explored the relationship between distortions in signal intensity following TwoRA and the original intensity level prior to TwoRA. The fact that the intensity range is limited by background noise on one end and saturation on the other end implies that intensity may only be shifted by a limited amount. This relationship bears important consequences on the con- sistency of the TwoRA protocol in amplifying targets with varying intensities across different samples. Thus, the shifts in intensity following amplification will not appear to be equivalent in the two samples if the shift in one of the samples is limited by the range of the scanner. This has Table 3: Similarity in significance calls from the OneRA and the TwoRA (DRG,SN) comparisons using the Z-scores and limma statistics. Number of top significant genes Z-scores SSS(%) FDR SSS (%) FDR 100 300 500 59 59 63 OneRA TwoRA 3.7E-3 7.1E-2 0.201 1.1E-2 0.11 0.214 87 87 87 The significance similarity score (SSS) is the percentage of genes common to the OneRA and the TwoRA selected sets of top significant genes. Limma OneRA 5.23E-14 2.08E-12 1.52E-11 TwoRA 1.72E-14 6.56E-13 5.59E-12 Page 11 of 14 (page number not for citation purposes)