BMC Genomics 2006, 7:252 http://www.biomedcentral.com/1471-2164/7/252 and although laser microdissection provides a solution to the problem of tissue procurement, the small amount of RNA that can be harvested has precluded a straightfor- ward combination of both technologies. This limitation is compounded by the need of replication essential for sta- tistical analysis. Another scenario in which the lack of sufficient tissue availability has been challenging is the correlation of the phenotype in individual experimental animals with com- prehensive gene expression profiles. For example, so far it has not been possible to correlate the inter-individual behavioural variability in animal models of chronic pain with the corresponding correlates in gene expression in the principal anatomical components of the pain pathway as such structures in individual animals do not yield suffi- cient amounts of RNA for standard hybridization proto- cols. To overcome these issues, increasingly sophisticated approaches to RNA amplification from small tissue sam- ples have been developed for use with microarrays. These fall principally into two categories. One is based on PCR and is characterized by an exponential increase in copy number while the other is based on the T7-polymerase in- vitro transcription (IVT) to achieve a linear amplification of targets. For maximum fidelity, linearity of target ampli- fication is desirable. Thus, substantial work has focused on exploring the T7 linear techniques [1-4], in particular the Affymetrix small sample protocol II has been assessed by numerous studies with interesting results. Analysis based on correlating intensity levels and assessing con- cordance in detection calls has indicated a high level of reproducibility [2,3,5,6]. However, occasional failure to maintain the true abundance level of transcripts was also found [3,5,6] due to the protocol 3'bias [1,2,4,6,7]. Such bias is thought to be related to the use of random hexam- ers to prime the RT reaction in the additional round of amplification. With priming that is remote from the 3'end, RT may not be successfully completed causing a diminution in the signal from the 5' regions. These observations are useful indicators of protocol valid- ity; though, the ultimate fidelity criterion is the ability to maintain differential expression between different tissues or under varying experimental conditions. Some previous studies have reported a 50% drop in significant changes in gene expression using RNA amplification [4], suggesting that RNA amplification may suffer major problems and is potentially unsuitable for microarray analysis. In this study, we critically appraise the suitability and mer- its of transcript amplification from small biological sam- ples for microarray expression profiling using the Affymetrix small sample protocol II. We confirm previous findings on the reproducibility and fidelity of the proto- col. We present compelling evidence for the 3' bias intro- duced by RNA amplification. We show how distortions in intensity may be reflected in the expression ratios from biologically different samples. Importantly, we explore the effect of distortions in expression ratios on their statis- tical significance. Results In this study, we undertake a detailed analysis of target amplification for microarrays using the Affymetrix small sample protocol II. This analysis was performed using control data from standard protocol preparations as refer- ence. For the rest of the article, we refer to the standard protocol as the OneRA (one round amplification) proto- col and the small sample protocol as the TwoRA (two rounds amplification) protocol. TwoRA and OneRA sam- ples from three different biological groups were used in this study: the DRG (dorsal root ganglia) group, the SN (spinal cord from normal animals) group and the SA (spi- nal cord from animals with axotomised sciatic nerve) group. In each group, there are 4 replicates from the TwoRA and 3 replicates from the OneRA (Figure 1a). While, the main objective of this study is to assess the extent to which biologically relevant variation in gene expression can be detected in the TwoRA, we begin by assessing the reproducibility of the TwoRA protocol and its fidelity in maintaining the relative abundance of tar- gets. Reproducibility and fidelity in maintaining expression levels Scatter plots of log2 intensities from paired TwoRA repli- cates show expectedly high level of consistency similar to that observed with the OneRA replicates (Figure 1b &1c) ((r) ranging from 0.990 to 0.994 among all possible pairs of the TwoRA replicates and the OneRA replicates from all biological groups). However, comparing the average log2 intensity values from the OneRA and the TwoRA (Figure 1d) for a single tissue, we see evidence of variability implying that the TwoRA protocol may distort the signal. We used an ANOVA approach to confirm that the variabil- ity between protocol groups is greater than that among replicates within each group. In particular, a one-way, two-levels ANOVA analysis was performed for each gene separately, with 3 measurements from the OneRA (level 1) and 4 measurements from the TwoRA (level 2). First, the between group mean sum of squares MSA as well as the mean residual sum of squares MSE were calculated. The median of the MSA was higher than the median of the MSE (given in parenthesis) in all biological groups: DRG 0.050 (0.023), SN 0.062 (0.016), SA 0.068. Page 2 of 14 (page number not for citation purposes)