Adomas et al. BMC Genomics 2010, 11:477 http://www.biomedcentral.com/1471-2164/11/477 Page 3 of 16 Table 1 Overview of the results comparing use of oligo(dT) and multi-targeted primers (MTPs) for reverse transcription Feature Number of well measured genes1 on the array Number of genes significantly differentially expressed (P ≤ 0.05) Number of up-regulated genes (P ≤ 0.05) Number of down-regulated genes (P ≤ 0.05) Highest gene expression ratio Average gene expression ratio GEL502 Number of genes with gene expression ratio greater or equal to GEL50 (P ≤ 0.05) Saccharomyces cerevisiae Nitrogen depletion Neurospora crassa Protoperithecial developmenr Oligo (dT) MTP Common Oligo (dT) MTP Common 4620 6042 2155 3577 1080 1855 1075 1722 49.15 32.6 2.05 1.77 1.51 1.27 1237 2617 4573 1839 925 912 1166 469 172 66 106 24.26 1.96 1.91 102 406 232 174 10.74 1.43 2.08 141 416 2021 31 61 52 1 well measured genes recorded after data normalization (foreground fluorescence signals were at least three standard deviations of the distribution of intensities of the background pixels for that gene). 2 GEL50 - gene expression level at which there is a 50% empirical probability of a significant call. without a slope with regard to number of MTP binding sites (P < 0.0001, slope 0.05). However, a model featur- ing the number of MTP binding sites is also a statisti- cally significant predictor of the probability of a statistically significant call when oligo-dT alone is used (P = 0.02, slope 0.03). More saliently, use of the MTP increased the number of genes that were identified as significantly differentially expressed by 66%. We analyzed the frequency of MTP priming sites among genes identified as significantly differentially expressed in nitrogen-depleted yeast. Out of 1738 genes indicated by MTPs exclusively as differentially expressed, 342 did not have an MTP recognition site and 1396 genes contained one to 29 priming sites. Among 316 genes exclusively identified by oligo-dT pri- mers as significantly differentially expressed in response to nitrogen depletion, 68 did not contain an MTP recognition site and 248 exhibited one to 18 priming sites. For the genes with the highest number of priming sites, the gene expression levels calculated based on the MTP experiment were similar to the oligo-dT experi- ment and typically yielded ratios close to one, indicating that a higher number of exact MTP priming sites per se did not increase detection of significant gene expression differences. The ratio of genes abundantly expressed to genes meagerly expressed was largely unchanged when MTPs were used (Table 1). Just two genes manifested a statisti- cally significant opposite direction of differential expres- sion in the MTP-microarray dataset compared to both the oligo-dT dataset and the independent RNA-Seq datasets (YDR179W-A, YKL203C). Out of 3577 genes detected as significantly differentially expressed by MTP-microarray, this is a very small number and may be reasonably attributed to experimental error in the MTP-primed reverse transcription and hybridizations. The highest and average changes in gene expression level were lower for MTP than for oligo-dT (Table 1). Lastly, the gene expression level at which there was a 50% empirical probability of a significant call (GEL50), illustrating the statistical power of the experiment, demonstrated greater resolution for MTP than for oligo- dT primers (Table 1). Three times more genes were detected as differentially expressed in the MTP experi- ment above its GEL50 threshold than were differentially expressed above the GEL50 threshold of the experiment performed with oligo-dT primers alone (Table 1). Functional classification of genes differentially expressed by S. cerevisiae grown in nitrogen-poor conditions While meager expression in nitrogen-poor medium was typical for genes functioning in protein synthesis and transcription, abundant expression was typical for genes coding for proteins involved in energy and genes of unknown function (Figure 1a). This result was obtained both with the aid of MTPs and with oligo-dT primers. Using oligo-dT primers, functional categories of metabolism, development, and energy were identi- fied as significantly affected under nitrogen deficiency (Fisher’s Exact test, P = 0.01, P = 2.3 × 10-21, P = 0.01). The MTP experiment identified the same functional categories, and provided additional experimental power, also identifying genes involved in regulation with the environment (P = 4.9 × 10-5). The classifica- tion for which the highest number of genes was indi- cated as significantly differentially expressed by either approach was metabolism. Within metabolism, the greater power of the MTP experiment provided a stronger signal of subcategory, as more measurements were statistically significant: 39% as indicated by MTP and 15% as indicated by oligo-dT primers were involved in amino acid metabolism; 36% and 15%, respectively, in carbohydrate metabolism; and 16% and 6% in lipid metabolism.