Adomas et al. BMC Genomics 2010, 11:477 http://www.biomedcentral.com/1471-2164/11/477 Page 4 of 16 Figure 1 Functional classification of genes significantly differentially expressed (P ≤ 0.05) by a) S. cerevisiae grown in nitrogen-poor conditions and b) N. crassa protoperithecia identified by transcript profiling using oligo(dT) (red) or multi-targeted primers (blue). Genes identified by both methods marked in green. Light-colored bars represent genes meagerly expressed and dark-colored bars represent genes abundantly expressed. The categories are sorted by the proportion of genes meagerly- to abundantly- expressed. MTPs were not biased for inference in particular path- ways or processes, providing an even increase in power across all of them. For nearly every pathway or process, the microarrays performed with MTPs resulted in a more complete assessment of expression of proteins/ enzymes building a pathway, assigning expression to more genes than the oligo-dT-primed experiment in each process (Table 2; see Additional File 3). Most of the genes coding for enzymes belonging to 26 pathways specifically related to nitrogen and amino acid metabo- lism and biosynthesis were meagerly expressed by yeast growing in nitrogen-poor conditions, e.g. glutamate metabolism and urea cycle (Table 2, Figures 2, 3). An opposite tendency of abundant expression in nitrogen- poor conditions was observed among genes encoding proteins composing energy-related pathways, e.g. the citrate cycle (Figures 1 &4). Typically, enzymes catalyz- ing reactions occurring in opposite directions exhibited opposite regulation (Figure 2). Furthermore, we were able to more reliably characterize the expression of mul- tiple genes coding for isozymes, isoforms with different cellular location and enzyme subunits (Figures 2, 3, 4). A total of 561 genes identified as differentially expressed by the MTP experiment and 364 genes identified as differ- entially expressed by the oligo-dT experiment belonged to environmental stress response genes that were inferred by evaluating the transcriptional responses to a wide range of stress stimuli, including nitrogen depletion [25]. RNA sequencing for assessment of transcript abundance in S. cerevisiae under nitrogen deficiency To validate the microarray profiling results, we performed RNA tag sequencing with a modified Digital Gene Expres- sion protocol that facilitated use of our custom oligonu- cleotide primers. We sequenced 6.0 × 105 16-17bp tags for yeast grown in nitrogen-rich conditions, primed with oligo-dT, out of which 2.7 × 105 exactly matched a single mRNA sequence in the S. cerevisiae genome. Similarly, we sequenced 1.3 × 106 tags from nitrogen deprived yeast, primed with oligo-dT, including 5.7 × 105 tags with an exact single match. We sequenced 3.8 × 106 and 1.3 × 106 tags, respectively, from yeast grown in nitrogen-rich condi- tions that had been MTP-primed; and 3.1 × 106 and 1.4 × 106, respectively, for yeast deprived of nitrogen that had been MTP-primed. The MTP-primed sequencing yielded larger numbers of reads in all categories. The proportion of reads mapping to rRNAs and tRNAs was slightly increased with MTPs, but with both oligo-dT and MTP priming this proportion was negligible (<0.01% of all single matches, Table 3). MTPs also did not prime a less