Adomas et al. BMC Genomics 2010, 11:477 http://www.biomedcentral.com/1471-2164/11/477 affected in our study may be related to growth arrest or to a morphological switch from the yeast to a filamen- tous or pseudohyphal growth triggered by nitrogen star- vation in the presence of a fermentable carbon source, such as glucose [41]. Nutrient and nitrogen sensing plays important roles in fungal development in general, and specifically in critical aspects of pathogenicity and virulence, for both animal and plant pathogens. Dimorphic pathogens such as the phytopathogenic smut fungi, Ustilago maydis and Microbotryum violaceum, must switch from a yeast-like to a filamentous form in order to cause disease [42]. N. crassa protoperithecia consist of a small knot of vegetative hyphae surrounding ascogonial cells. Their formation requires extensive cell proliferation. Thus, it was not surprising to find a large proportion of differ- entially expressed genes involved in metabolism, espe- cially carbohydrate metabolism (Table 2), providing energy and the robust structural components necessary for specialized cell formation. The shift in transcript accumulation of genes involved in fatty acid metabo- lism (Figure 5) supports previous observations that the fatty acid composition of sexual tissues of Neurospora differ substantially from the composition of asexual tis- sues. For instance, mutations in the gene encoding the b-fatty acid synthase affect sexual development [43]. On the other hand, we expected to identify known developmental genes responsible for the formation of protoperithecial reproductive structures, but the num- ber of genes significantly differentially expressed involved in the functional category of development was very low. One of them coded for clock controlled pheromone ccg-4 precursor which had been first iden- tified as a gene that is expressed with a 22 h rhythm under the control of the circadian biological clock [44]. The expression of the pheromone precursor genes is mating-type specific and is under the control of the mating type locus. These genes are highly expressed in conidia and under conditions that favor sexual development [45]. We found a reduced level of transcripts related to pro- tein synthesis (Figure 1, see Additional File 7) in both S. cerevisiae under nitrogen starvation and in N. crassa sexual development occurring as a developmental response to nitrogen depletion. Ribosome biogenesis and protein translation are among the most energy-con- suming cellular processes [46]. Thus, these pathways are tightly controlled upon nutrient limitation. Decreasing levels of protein, RNA and soluble aminonitrogen were observed during protoperithecia development as early as 1975 [47]. Additionally, ribosomes are rapidly degraded by autophagy upon nutrient starvation in S. cerevisiae, implying that degradation of excessive ribosomes may help to shut down protein translation rapidly and Page 12 of 16 provide an important source of new building blocks to maintain cellular homeostasis [48]. The MTPs used in this study were designed to prime 76-85% of mRNAs rather than all the genes, so that reverse transcription of rRNA and tRNA could be simultaneously minimized. An additional step of primer design could be conducted to specifically target the remaining sequences to eliminate or further diminish the need to rely on low-complexity, low-specificity oligo-dT primers. For transcript profiling in prokaryotes, such a tiered approach would constitute an appealing alternative to the use of non-specific random primers. Correspondingly, the modification of Illumina massively parallel RNA tag sequencing presented here, involving the use of custom primers, presents the possibility of performing Digital Gene Expression assays on transcrip- tomes lacking poly(A) tails or on a designable fraction of all the genes. Furthermore, future applications of multi-targeted primers may include concurrent selective amplification of DNA sequences of interest from one or multiple genomes for the high-throughput sequencing of multiple homologous loci. Locked nucleic acid nucleotides could be included in the MTP sequence to increase binding strength and specificity, and to decrease non-specific amplification [49]. Conclusions Here we presented a novel priming strategy for genome- wide gene expression assays, featuring selective targeting of mRNA and counter-selection against rRNA and tRNA. We demonstrated superior performance of two MTPs compared to oligo-dT by microarray profiling of the response of Saccharomyces cerevisiae to nitrogen deficiency and by profiling Neurospora crassa early sex- ual development. MTPs resulted in higher sensitivity, yielding more well measured genes after data normaliza- tion, more genes significantly differentially expressed, and a greater power to detect meager differences in gene expression. Our results provide the most complete and detailed expression profiles of the nitrogen starva- tion response to date. Future applications of multi-tar- geted primers may include concurrent selective amplification of DNA sequences of interest from one or multiple genomes for the high-throughput sequencing of multiple homologous loci. Methods Multi-targeted primers (MTPs) Sequences for all three types of RNA were obtained from the Saccharomyces Genome Database [50] and the Neurospora crassa Genome Database [35]. To identify a degenerate sequence that occurs in a maximal number of mRNAs and in a minimal number of tRNAs and rRNAs, exhaustive search is not a feasible option as