BMC Genomics 2006, 7:252 http://www.biomedcentral.com/1471-2164/7/252 nEFififgiecucatrneocef7variation in expression ratios on their statistical sig- Effect of variation in expression ratios on their statis- tical significance. Scatter plots of FDR corrected nlPv (negated log transformed p-value) from the Limma analysis of the OneRA and the TwoRA DRG and SN samples. As a result of negating the p-values, large nlPv indicate stronger evidence of differential expression. Data points in red repre- sent the FCE probesets. The dashed line is at nlPv = 10 in the TwoRA, above which genes may be considered significant. the consequence of distorting the expression ratios, as clearly demonstrated by our data. Unsurprisingly, the statistical significance of expression ratios is only affected when the expression ratio in the TwoRA is reduced to the point where it can no longer be distinguished from noise. Importantly, large ratios are less likely to be critically diminished and more likely to remain significant following TwoRA. This explains why despite the distortions in ratios in our dataset, there was up to 87% agreement in the top significant genes (nlPv > 20) from the TwoRA and OneRA (DRG,SN). On the other hand, less agreement was observed among the less pro- nounced ratios (69%) since distortions are more critical. This leads us to the important conclusion that TwoRA may affect the statistical significance of genes with moder- ate expression ratios to a greater extent. Another impor- tant finding by this study is that limma performs best at recognising similarity in differential expression between the OneRA and the TwoRA. Thus, the choice of statistics will also affect the quality of information obtained from microarray analysis of amplified samples. Conclusion We conclude that the Affymetix small sample amplifica- tion protocol is useful with the following caveats: First, it should be only used when tissue homogeneity is a crucial factor and sufficient amounts of starting material cannot be obtained by any other means. Secondly, target amplifi- cation using the small sample protocol appears to be suit- able in situations where big differences in gene expression are expected. Fortunately, it is reasonable to expect large differential expressions with experiments characterising different cells within a mixed tissue where amplification of transcript is necessary. Finally, application of such tech- nique in combination with limma statistical analysis is useful in experiments where the ultimate purpose is to experimentally characterise a finite number of targets, for example the top 100 differentially expressed genes. However, expression data obtained from amplified sam- ples might be less suitable for more comprehensive numerical analysis, for example characterising regulatory networks, due to the problems caused by possible shifts in signal and expression ratios. Methods - Tissue isolation and RNA extraction Three pools of tissue were prepared from C57/B6 adult male mice after cervical dislocation or decapitation. Tissue from each animal was dissected within 10 minutes of kill- ing and collected on dry ice. One sample consisted of dor- sal root ganglia (DRG) of all spinal levels from 10 naïve mice. Another sample (SA) was the lumbar enlargement of the spinal cord of 5 animals that had undergone a uni- lateral sciatic nerve transection at mid-thigh level under isoflurane anesthesia 7 days previously. A third sample (SN) was the lumbar enlargement of the spinal cord of 5 normal mice. RNA was prepared using the Trizol method (Invitrogen) followed by a cleanup step using the RNAe- asy Mini kit (Qiagen). RNA quality and quantity was measured by microfluidic electrophoresis (Nanochip run- ning on a 2100 Bioanalyzer, Agilent Technologies, Palo Alto, USA). The 28S/18S RNA ranged from 1.3 to 1.4 in all samples. The total amount of RNA was 42.2 μg, 40.1 μg, and 25.3 μg for the DRG, SN, SA samples respectively. The corresponding RNA integrity numbers (RINs) were 8.3, 9.3 and 9.4. RINs larger than 8 are considered to be perfect for downstream applications [37]. - RNA amplification and chip hybridisation RNA processing and hybridization followed the Affyme- trix methodology. In brief, the Affymetrix standard proto- col [8] (in the article referred to as the OneRA protocol) was used to generate three labelled cRNA samples from each tissue pool using 5 μg of total RNA as starting mate- rial (Figure 1a). The Affymetrix small sample protocol ver- sion 2 [9] (referred to as the TwoRA protocol) that Page 12 of 14 (page number not for citation purposes)