Adomas et al. BMC Genomics 2010, 11:477 http://www.biomedcentral.com/1471-2164/11/477 that were detected by oligo-dT alone) than did oligo-dT priming alone (368 not detected by oligo-dT that were detected by MTP). Correlations between replicate tag sequencing results and between replicate MTP-based microarray results were similar. Next generation RNA tag sequencing offers a promise of increasing power, depth, and sensitivity of gene expression analysis, but even a limited depth of RNA sequencing remains costly compared to a highly replicated experiment using micro- arrays generated in the lab. Finally, qRT-PCR validated the results based on MTP- priming. It confirmed that genes identified as well mea- sured or as significantly differentially expressed only by MTP-primed microarray, had changed in expression in the direction revealed by the MTP-primed experiments, regardless of the primer combination used. This obser- vation suggests that incorporating MTPs increases the power for detection of differential expression by micro- arrays, by increasing the coverage of the priming of reverse transcription while maintaining low noise due to nonspecific priming. Logistic regressions showed some evidence that genes with more priming sites were mea- sured with increased power with MTPs applied than genes with fewer MTP priming sites, but this observa- tion was true (albeit to a lesser extent) for oligo-dT priming as well. This commonality may well arise because longer genes have more MTP sites and are bet- ter measured with either MTPs or oligo-dT primers. Considering the 4762 observations underlying these effects, their statistical significance is unsurprising. The effect sizes are not large. It would be possible to proce- durally minimize the variation in the number of MTP sites, but effectively, this minimization of variation would entail searching for MTPs that were less powerful for some genes. That would be counter-productive to the effort to measure genome-wide gene expression for as many genes as possible to the best precision possible. Because such gene-specific effects exist at approximately the same levels no matter the method of priming, decent statistical approaches toward microarray data have featured effects estimated for each gene, thus pro- viding P-values and confidence or credible intervals that appropriately reflect the variation in power across genes. Markedly successful enhancement of the power of tran- scriptomic approaches was achieved here by usage of MTPs subsequent to a crude filtration provided by poly- A+ purification columns, which typically bring the RNA content to approximately 50% poly-A+ RNA and 50% other RNA [32,33]. An alternate approach with potential to further increase power would be to forego any col- umn purification of poly-A+, relying solely on MTPs to pick out messenger RNA from other species of RNA. Functionally, inclusion of MTPs provided a more complete picture of cellular processes undergoing Page 11 of 16 change during fungal growth in nitrogen deficient con- ditions and in early sexual development. Interestingly, the most striking difference in the ability to detect genes differentially expressed between MTPs and oligo-dT pri- mers was observed among N. crassa unknown and unclassified genes (Figure 1b). Genes expressed at a low level have a lower probability of being identified and characterized than those expressed at a higher level [34]. Only 42% of genes in N. crassa genome have been assigned known functions [35] so identifying their expression signatures and inferring functional classifica- tion are key objectives of future experimentation [36]. The increased levels of yeast transcripts coding for enzymes involved in the TCA cycle observed here (Figure 4) supports a finding that the content of TCA cycle compounds increases during yeast starvation [37]. Both carbon and nitrogen limitation have been shown to enhance respiration relative to fermentation in yeast [21,38]. Amino acid metabolism was strongly affected by exposure to nitrogen deficiency, as in the filamentous fungus Magnaporthe grisea [17]. Prevalent down-regula- tion of glutamate metabolism pathway (Figure 2) was in accordance with decline of total glutathione pool found in nitrogen-starved S. cerevisiae [39]. Similarly, the urea cycle was down-regulated, with an exception of the branch responsible for urea degradation (Figure 3). Urea amidolyase, the enzyme corresponding to that branch, contains both urea carboxylase and allophanate hydro- lase activities. It also converts allantoin to ammonia and carbon dioxide, enabling S. cerevisiae to use allantoin as a sole nitrogen source [40]. The urea cycle operates in terrestrial animals to detoxify ammonia, and in yeast plays a principal role in the biosynthesis of arginine. Within these pathways, we were able to characterize expression of multiple isozymes and isoforms (Figures 2, 3, 4) providing a key component of a full understanding of expression regulation in nitrogen-deficient medium. The general trend of shutting down amino acid biosynth- esis and metabolism appears essential for survival during prolonged periods of starvation. These results from our experiments are entirely consistent with a functional category analysis of independent data from the unrepli- cated time-course experiment by Gasch et al. [25]. A number of genes abundantly expressed by yeast during growth in nitrogen-poor conditions are compo- nents of the environmental stress response that was characterized by evaluating the transcriptional responses to a wide range of stress stimuli, including nitrogen depletion [25]. The remaining genes might be involved in immediate responses specific to nitrogen limitation, but many are likely associated with transitive effects that occur over time, such as successive nutrient depletion, pH change, cell density, and growth arrest. The finding that development-related genes were significantly